Stem Cell Niche

Methods in
Molecular Biology 1035
Kursad Turksen Editor
Stem Cell
Niche
Methods and Protocols
METHODS IN M O L E C U L A R B I O LO G Y ™
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
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Stem Cell Niche
Methods and Protocols
Edited by
Kursad Turksen
Sprott Centre for Stem Cell Research, Regenerative Medicine Program,
Ottawa Hospital Research Institute, Toronto, ON, Canada
Editor
Kursad Turksen
Sprott Centre for Stem Cell Research
Regenerative Medicine Program
Ottawa Hospital Research Institute
Toronto, ON, Canada
ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-62703-507-1 ISBN 978-1-62703-508-8 (eBook)
DOI 10.1007/978-1-62703-508-8
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Preface
The idea that there was a specialized microenvironment or “niche” regulating hemopoietic
stem cell function was first proposed by Ray Schofield over three decades ago. As interest
in stem cell biology has exploded over the last 10 years, so too has the interest in the stem
cell niche. This explosion of interest and, more recently, cellular-molecular-biochemical
characterization of not only the hemopoietic stem cell niche but the niches for other stem
cells was the driving force for putting together a volume of protocols for investigating stem
cell niches. As always, it is not possible to collect all the different protocols in one volume;
however, I have attempted to select a subset of representative protocols that hopefully will
provide both a flavor of the field and hopefully stimulate new approaches and methodolo-
gies by those interested in the stem cell niche.
The protocols gathered here are faithful to the mission statement of the Methods in
Molecular Biology series: They are well established and described in an easy-to-follow step-
by-step fashion so as to be valuable for not only experts but also novices in the stem cell
field. That goal is achieved because of the generosity of the contributors who have carefully
described their protocols in this volume, and I am grateful for their efforts.
My thanks as well go to Dr. John Walker, the Editor in Chief of the Methods in
Molecular Biology series, for giving me the opportunity to create this volume and for sup-
porting me along the way.
I am also grateful to Patrick Marton, the Editor of Methods in Molecular Biology and
Springer’s Protocol series, for his continuous support from idea to completion of this
volume.
Finally, I would like to thank Tamara Cabrero for her outstanding editorial work during
the production of this volume.
Toronto, ON, Canada Kursad Turksen
v
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Immunostaining of Germline Stem Cells and the Niche

in Drosophila Ovaries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Lichao Luo, Phing Chian Chai, and Yu Cai
2 Genetic, Immunofluorescence Labeling, and In Situ Hybridization
Techniques in Identification of Stem Cells in Male and Female
Germline Niches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Shree Ram Singh, Ying Liu, Madhuri Kango-Singh, and Eviatar Nevo
3 Visualization of Adult Stem Cells Within Their Niches Using
the Drosophila Germline as a Model System. . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Annekatrin König and Halyna R. Shcherbata
4 Morphometric Evaluation of the Spermatogonial Stem Cell Distribution
and Niche in Vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Paulo Henrique Almeida Campos-Junior, Guilherme Mattos Jardim Costa,
Gleide Fernandes de Avelar, Tânia Mara Segatelli, Samyra Maria Santos
Nassif Lacerda, Pedro Manuel Aponte, and Luiz Renato de França
5 In Vitro Construction of 2D and 3D Simulations of the Murine
Hematopoietic Niche . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Brahmananda Reddy Chitteti, Monique Bethel, Sherry L. Voytik-Harbin,
Melissa A. Kacena, and Edward F. Srour
6 Isolation of Embryonic Hematopoietic Niche Cells by Flow Cytometry
and Laser Capture Microdissection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Daisuke Sugiyama and Tatsuya Sasaki
7 Isolation and Enrichment of Stro-1 Immunoselected Mesenchymal
Stem Cells from Adult Human Bone Marrow . . . . . . . . . . . . . . . . . . . . . . . . . 67
Emma L. Williams, Kate White, and Richard O.C. Oreffo
8 Primary Marrow-Derived Stromal Cells: Isolation and Manipulation . . . . . . . . 75
Aravind Ramakrishnan, Beverly Torok-Storb, and Manoj M. Pillai
9 Detection In Vitro and Quantitative Estimation of Artificial
Microterritories Which Promote Osteogenic Differentiation and Maturation
of Stromal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Igor A. Khlusov, Natalya M. Shevtsova, and Marina Yu. Khlusova
10 The Prospective Isolation of Viable, High Ploidy Megakaryocytes
from Adult Murine Bone Marrow by Fluorescence Activated Cell Sorting . . . . 121
vii
viii Contents
Shen Y. Heazlewood, Brenda Williams, Melonie J. Storan,

and Susan K. Nilsson
11 Looking for the Niche: Substance Delivery into the Lateral Ventricle
of the Brain: The Osmotic Minipump System . . . . . . . . . . . . . . . . . . . . . . . . . 135
María Victoria Gómez Gaviro, Pedro Luis Sánchez Fernández,
Robin Lovell Badge, and Francisco Fernández Avilés
12 Unbiased Stereological Method to Assess Proliferation Throughout
the Subependymal Zone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Ana Mendanha Falcão, Joana Almeida Palha, Ana Catarina Ferreira,
Fernanda Marques, Nuno Sousa, and João Carlos Sousa
13 Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation
of Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Paras Kumar Mishra, Nicholas John Kuypers, Shree Ram Singh,
Noel Diaz Leiberh, Vishalakshi Chavali, and Suresh C. Tyagi
14 Human and Murine Skeletal Muscle Reserve Cells . . . . . . . . . . . . . . . . . . . . . 165
Rana Abou-Khalil, Fabien Le Grand, and Bénédicte Chazaud
15 Modulation of the Host Skeletal Muscle Niche for Donor Satellite
Cell Grafting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Luisa Boldrin and Jennifer E. Morgan
16 Isolation of c-Kit+ Human Amniotic Fluid Stem Cells from Second Trimester . . . 191
Michela Pozzobon, Martina Piccoli, Andrea Alex Schiavo,
Anthony Atala, and Paolo De Coppi
17 Hypoxia and Visualization of the Stem Cell Niche. . . . . . . . . . . . . . . . . . . . . . 199
Ali Dalloul
18 Detection and Isolation of Human Disseminated Tumor Cells
in the Murine Bone Marrow Stem Cell Niche . . . . . . . . . . . . . . . . . . . . . . . . . 207
Yusuke Shiozawa, Russell S. Taichman, and Evan T. Keller
19 Identification and Separation of Normal Hematopoietic Stem Cells
and Leukemia Stem Cells from Patients with Acute Myeloid Leukemia . . . . . . 217
Van T. Hoang, Isabel Hoffmann, Karina Borowski,
Abraham Zepeda-Moreno, Dan Ran, Eike C. Buss,
Patrick Wuchter, Volker Eckstein, and Anthony D. Ho
20 Serial Orthotopic Transplantation of Epithelial Tumors
in Single-Cell Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Heather A. McCauley and Géraldine Guasch
21 Isolation and Propagation of Colon Cancer Stem Cells . . . . . . . . . . . . . . . . . . 247
Pramudita R. Prasetyanti, Cheryl Zimberlin, Felipe De Sousa E. Melo,
and Jan Paul Medema
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Contributors
RANA ABOU-KHALIL • INSERM U781, Hôpital Necker-Enfants Malades, Paris, France

JOANA ALMEIDA PALHA • Life and Health Sciences Research Institute (ICVS),
School of Health Sciences, University of Minho, Braga, Portugal
PEDRO MANUEL APONTE • Laboratory of Cellular Biology, Department of Morphology,
Institute of Biological Sciences, Federal University of Minas Gerais,
Belo Horizonte, Brazil
ANTHONY ATALA • Wake Forest Institute for Regenerative Medicine,
Wake Forest University School of Medicine, Winston-Salem, NC, USA;
Department of Vascular and Endovascular Surgery, Wake Forest University School
of Medicine, Winston-Salem, NC, USA
GLEIDE FERNANDES DE AVELAR • Laboratory of Cellular Biology, Department of Morphology,
FRANCISCO FERNÁNDEZ AVILÉS • Servicio de Cardiología, Instituto de Investigación
Sanitaria Hospital Gregorio Marañón, Madrid, Spain
ROBIN LOVELL BADGE • Division of Stem Cell Biology and Developmental Genetics,
National Institute for Medical Research, MRC, London, UK
MONIQUE BETHEL • Orthopaedic Surgery, Indiana University School of Medicine,
Indianapolis, IN, USA
LUISA BOLDRIN • The Dubowitz Neuromuscular Centre UCL, Institute of Child Health,
London, UK
KARINA BOROWSKI • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
EIKE C. BUSS • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
YU CAI • Temasek Life Sciences Laboratory, Singapore; Department of Biological Sciences,
National University of Singapore, Singapore
PHING CHIAN CHAI • Temasek Life Sciences Laboratory, Singapore; Department of
Biological Sciences, National University of Singapore, Singapore
VISHALAKSHI CHAVALI • University of Nebraska Medical Center, Omaha, NE, USA
BÉNÉDICTE CHAZAUD • Institut Cochin, INSERM U1016, Paris, France
BRAHMANANDA REDDY CHITTETI • Division of Hematology-Oncology,
Indiana University School of Medicine, Indianapolis, IN, USA
PAOLO DE COPPI • Department of Woman and Child Health and Pediatric Surgery,
University of Padova, Padova, Italy; Surgery Unit, Institute of Child Health,
Great Ormond Street Hospital, University College, London, UK
ix
x Contributors
GUILHERME MATTOS JARDIM COSTA • Laboratory of Cellular Biology,

Department of Morphology, Institute of Biological Sciences, Federal University
of Minas Gerais, Belo Horizonte, Brazil
ALI DALLOUL • Department of Cell Biology, Nancy Medical School,
Université de Lorraine, Nancy, France
NOEL DIAZ LEIBERH • Department of Physiology & Biophysics, School of Medicine,
University of Louisville, Louisville, KY, USA
VOLKER ECKSTEIN • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
ANA MENDANHA FALCÃO • Life and Health Sciences Research Institute (ICVS),
PEDRO LUIS SÁNCHEZ FERNÁNDEZ • Servicio de Cardiología, Instituto de Investigación
Sanitaria Hospital Gregorio Marañón, Madrid, Spain
ANA CATARINA FERREIRA • Life and Health Sciences Research Institute (ICVS),
LUIZ RENATO DE FRANÇA • Laboratory of Cellular Biology, Department of Morphology,
MARÍA VICTORIA GÓMEZ GAVIRO • Servicio de Cardiología, Instituto de
Investigación Sanitaria Hospital Gregorio Marañón, Madrid, Spain
FABIEN LE GRAND • Institut Cochin, INSERM U1016, Paris, France
GÉRALDINE GUASCH • Division of Developmental Biology, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH, USA
SHEN Y. HEAZLEWOOD • Materials Science and Engineering, Commonwealth Scientific
and Industrial Research Organization, Melbourne, VIC, Australia
PAULO HENRIQUE ALMEIDA CAMPOS-JUNIOR • Laboratory of Cellular Biology,
Department of Morphology, Institute of Biological Sciences, Federal University of Minas
Gerais, Belo Horizonte, Brazil
ANTHONY D. HO • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
VAN T. HOANG • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
ISABEL HOFFMANN • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
MELISSA A. KACENA • Orthopaedic Surgery, Indiana University School of Medicine,
Indianapolis, IN, USA
MADHURI KANGO-SINGH • Department of Biology, Center for Tissue Regeneration
and Engineering at Dayton (TREND), University of Dayton, Dayton, OH, USA
EVAN T. KELLER • Departments of Urology, University of Michigan Medical School,
Ann Arbor, MI, USA
IGOR A. KHLUSOV • Scientific Educational Center, Biocompatible Materials
and Bioengineering, Siberian State Medical University, Tomsk, Russia
MARINA YU. KHLUSOVA • Scientific Educational Center, Biocompatible Materials
ANNEKATRIN KÖNIG • Max Planck Research Group of Gene Expression and Signaling,
Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
Contributors xi
NICHOLAS JOHN KUYPERS • Department of Anatomical Sciences and Neurobiology,

SAMYRA MARIA SANTOS NASSIF LACERDA • Laboratory of Cellular Biology, Department of
Morphology, Institute of Biological Sciences, Federal University of Minas Gerais,
YING LIU • Mouse Cancer Genetics Program, National Cancer Institute, NIH, Frederick,
MD, USA
LICHAO LUO • Temasek Life Sciences Laboratory, Singapore; Department of Biological
Sciences, National University of Singapore, Singapore
FERNANDA MARQUES • Life and Health Sciences Research Institute (ICVS),
HEATHER A. MCCAULEY • Division of Developmental Biology, Cincinnati Children’s
Hospital Medical Center, Cincinnati, OH, USA
JAN PAUL MEDEMA • Laboratory of Experimental Oncology and Radiobiology
Center for Experimental Molecular Medicine, Academic Medical Center,
Amsterdam, The Netherlands
FELIPE DE SOUSA E. MELO • Laboratory of Experimental Oncology and Radiobiology
PARAS KUMAR MISHRA • Department of Cellular & Integrative Physiology,
University of Nebraska Medical Center, Omaha, USA
JENNIFER E. MORGAN • The Dubowitz Neuromuscular Centre UCL, Institute of Child
Health, London, UK
EVIATAR NEVO • Institute of Evolution, University of Haifa, Haifa, Israel
SUSAN K. NILSSON • Materials Science and Engineering, Commonwealth Scientific
and Industrial Research Organization, Melbourne, Australia; Department of Anatomy
and Developmental Biology, Monash University, Melbourne, Australia
RICHARD O.C. OREFFO • Bone and Joint Research Group, University of Southampton
Medical School, Southampton, UK
MARTINA PICCOLI • Città della Speranza Foundation, Padova, Italy
MANOJ M. PILLAI • Division of Medical Oncology, University of Colorado Anschutz
Medical Campus, Aurora, CO, USA
MICHELA POZZOBON • Città della Speranza Foundation, Padova, Italy
PRAMUDITA R. PRASETYANTI • Laboratory of Experimental Oncology and Radiobiology
Center for Experimental Molecular Medicine, Academic Medical Center, Amsterdam,
The Netherlands
ARAVIND RAMAKRISHNAN • Clinical Research Division, Fred Hutchinson Cancer Research
Center, Seattle, WA, USA
DAN RAN • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
TATSUYA SASAKI • Division of Hematopoietic Stem Cells, Advanced Medical Initiatives,
Department of Advanced Medical Initiatives, Kyushu University Faculty of Medical
Sciences, Fukuoka, Japan
ANDREA ALEX SCHIAVO • Città della Speranza Foundation, Padova, Italy
TÂNIA MARA SEGATELLI • Laboratory of Cellular Biology, Department of Morphology,
xii Contributors
HALYNA R. SHCHERBATA • Max Planck Research Group of Gene Expression

and Signaling, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
NATALYA M. SHEVTSOVA • Scientific Educational Center, Biocompatible Materials
YUSUKE SHIOZAWA • Department of Periodontics and Oral Medicine,
University of Michigan School of Dentistry, Ann Arbor, MI, USA
SHREE RAM SINGH • Mouse Cancer Genetics Program, National Cancer Institute, NIH,
Frederick, MD, USA
JOÃO CARLOS SOUSA • Life and Health Sciences Research Institute (ICVS),
NUNO SOUSA • Life and Health Sciences Research Institute (ICVS),
EDWARD F. SROUR • Pediatrics/Herman B Wells Center for Pediatric Research,
Indiana University School of Medicine, Indianapolis, IN, USA
MELONIE J. STORAN • Materials Science and Engineering, Commonwealth Scientific
and Industrial Research Organization, Melbourne, Australia
DAISUKE SUGIYAMA • Division of Hematopoietic Stem Cells, Advanced Medical Initiatives,
Department of Advanced Medical Initiatives, Kyushu University
Faculty of Medical Sciences, Fukuoka, Japan
RUSSELL S. TAICHMAN • Department of Periodontics and Oral Medicine,
University of Michigan School of Dentistry, Ann Arbor, MI, USA
BEVERLY TOROK-STORB • Clinical Research Division, Fred Hutchinson Cancer
Research Center, Seattle, WA, USA
SURESH C. TYAGI • Department of Physiology & Biophysics, School of Medicine,
SHERRY L. VOYTIK-HARBIN • Purdue University Weldon School of Biomedical Engineering,
West Lafayette, IN, USA
KATE WHITE • Bone and Joint Research Group, University of Southampton Medical School,
Southampton, UK
BRENDA WILLIAMS • Materials Science and Engineering, Commonwealth Scientific
and Industrial Research Organization, Melbourne, Australia
EMMA L. WILLIAMS • Bone and Joint Research Group, University of Southampton Medical
School, Southampton, UK
PATRICK WUCHTER • Department of Internal Medicine V, University of Heidelberg,
Heidelberg, Germany
ABRAHAM ZEPEDA-MORENO • Department of Internal Medicine V,
University of Heidelberg, Heidelberg, Germany
CHERYL ZIMBERLIN • Laboratory of Experimental Oncology and Radiobiology
Chapter 1
Immunostaining of Germline Stem Cells and the Niche

in Drosophila Ovaries
Lichao Luo, Phing Chian Chai, and Yu Cai
Abstract
Stem cells have the ability to switch between proliferative (self-renewal) and differentiation modes. The
Drosophila germarium is a well-established in vivo model for the study of communication between stem cells
and their niche. One commonly used technique for such study is immunostaining that allows examination of
protein localization at a fixed time point. This chapter provides a detailed protocol for immunofluorescence
staining of Drosophila ovaries. This protocol has been optimized to enable explicit visualization of the niche
structure, as well as to maximize the degree of multiplexing for protein labeling and detection.
Key words Drosophila, Germline stem cells, Niche, Immunostaining
1 Introduction
For Drosophila germline stem cells (GSCs), the decision to undergo

proliferation or to differentiate is largely controlled by a cluster of
heterologous somatic cells surrounding the GSCs in a non-cell
autonomous fashion [1–3]. These somatic cells that constitute a
“niche” are composed of cap cells (CCs), terminal filament (TF)
cells, and escort cells (ECs) (Fig. 4) [4, 5]. Studies have shown that
molecular signals emanating from the niche, such as Decapentaplegic
(Dpp), as well as proper anchoring of the GSCs to the CCs (the
major signal-sending center), are essential to maintain GSC fate
[1, 5–7]. On the other hand, signals derived from the ECs play a
pivotal role in promoting GSC differentiation [8–10]. To elucidate
the mechanistic insight into how signals emanating from the niche
can affect GSC fate and to uncover novel niche signaling compo-
nents, it is useful to immuno-label proteins involved in the signal-
ing pathway in order to visualize their localization in both spatial
and temporal contexts.
One routinely used immuno-labeling techinique is immuno-
chemiluminescence, whose principle is to use an antibody-tagged
Kursad Turksen (ed.), Stem Cell Niche: Methods and Protocols, Methods in Molecular Biology, vol. 1035,
DOI 10.1007/978-1-62703-508-8_1, © Springer Science+Business Media, LLC 2013
1
2 Lichao Luo et al.
enzyme such as alkaline phosphatase (AP) or horseradish peroxi-

dase (HRP) to convert a chemical substrate to a light-emitting
product [11, 12]. Although this method is sensitive and has
broad dynamic range [13], the limited choice of chemiluminescence
compounds available restricts its practicability when simultane-
ous labeling of multiple proteins is needed. To study GSC and its
niche, we favor immunostaining because of its ability to multi-
plex. This is particularly important for simultaneous labeling of
multiple structures and signaling molecules within Drosophila
germarium. Here, we provide an effective immunostaining pro-
tocol to label GSCs and the niche in Drosophila ovaries. As the
niche at the anterior tip of the germarium is visually obstructed
by the muscle sheath, this protocol provides an easy process for
muscle sheath removal as to reveal a flatten structure of the ger-
marium. With that, coupled with our chosen markers, we are
able to distinguish GSCs and the niche even with a single fluo-
rescein. This would allow greater degree of multiplexing for the
detection of other signaling components.
2 Materials
2.1 10 M NaOH Weigh 4 g sodium hydroxide (NaOH) and dissolve them in 10 ml

sterile water.
2.2 PBS pH 7.4 Dissolve 7.6 g NaCl, 0.99 g Na2HPO4, and 0.41 g NaH2PO4·H2O
(1,000 ml) in 950 ml sterile water. Adjust the pH value to 7.4 using 10 M
NaOH (prepared in Subheading 2.1). Finally, bring the volume to
1,000 ml with sterile water.
2.3 PBT Add 1 ml Triton X-100 to 1,000 ml PBS (prepared in

Subheading 2.2) and mix.
2.4 20 % PFA Weigh 20 g paraformaldehyde powder, and then add 200 μl 10 M

NaOH (prepared in Subheading 2.1) and 40 ml PBS. Stir the mix-
ture at 60 °C until the paraformaldehyde is completely dissolved.
Add PBS to a final volume of 50 ml. Allow the solution to cool at
room temperature. Store the solution at 4 °C (see Note 1).
2.5 1 M HEPES Weigh 238.30 g HEPES and dissolve it in 900 ml sterile water. Use
10 M NaOH (prepared in Subheading 2.1) to adjust the pH to
7.5. Finally, bring the volume to 1,000 ml with sterile water.
2.6 Fixative Buffer Mix 700 μl PBS, 200 μl 20 % PFA, and 100 μl 1 M HEPES (see
Subheading 2.5) (see Notes 2 and 3).
2.7 3 % Bovine Weigh 30 g bovine serum albumin (BSA) and dissolve it in

Serum Albumin 1,000 ml PBT. Add 0.2 g sodium azide (NaN3) powder to prevent
microbial growth. Store the solution at 4 °C.
Immunostaining of Germline Stem Cells and the Niche in Drosophila Ovaries 3
Fig. 1 The needle used for dissection. Black arrow shows the tip of wolframium
wire that has been sharpened by electrolysis
2.8 Needle Insert a wolframium wire through the hole of a needle attached to a
syringe and secure one end of the wire with the plunger. The length
of the wire extending out of the needle can be adjusted by moving
the plunger. Sharpen the tip of the wire by electrolysis (Fig. 1).
2.9 Equipment Labquake (Model NO.415220, Barnstead international Inc.).

Nutating mixer (SER.#Q 002099, Labnet International Inc.).
Electrolysis machine(MP-1525, Shinseiki).
2.10 Microscope LEICA MZ12.5 stereomicroscope.

Zeiss LSM 510 META upright confocal microscope.
3 Methods
3.1 Fatten the Flies 1. Collect newly eclosed flies (within 24 h after eclosure) into
vials of fresh food with a dough of yeast paste and transfer
them daily into new vials with freshly prepared yeast paste (see
Note 4).
3.2 Dissecting All steps are performed at room temperature unless otherwise
and Staining stated. Steps 6–14 are carried out on a nutating mixer (see Note 5).
Take special care to ensure that the samples do not dry up all
the time.
1. Prepare a petri dish filled with PBS.
2. Anesthetize the flies on a carbon dioxide flowbed.
3. Use a forceps to hold the thorax of the anesthetized fly and
move it into the dish.
4. While grabbing the fly by one forceps, use another forceps to
tear the skin of the dorsal abdomen until the ovaries are visible
4 Lichao Luo et al.
Fig. 2 The schematic diagram of dissection
under the microscope. Remove all other parts of the fly body
(see Note 6, Fig. 2).
5. Repeat steps 3 and 4 until enough ovaries are collected and
immediately transfer the ovaries into a microfuge tube contain-
ing fixative buffer (see Note 7).
6. Fix the ovaries for 20 min (see Note 8).
7. Remove all fixative, and wash the ovaries in PBT for 5 min.
Repeat this washing step for another three times (see Note 9).
8. Remove PBT and block the ovaries in 3 % BSA for 30 min.
9. Remove the blocking buffer and add the primary antibodies
diluted in 3 % BSA.
10. To label the niche and stem cells, incubate the sample for 2–4 h
with the following primary antibodies: (a) mouse monoclonal
anti-α-Spectrin [3A9, 1:100, Developmental Studies
Hybridoma Bank(DSHB)] and (b) anti-Lamin C (LC28.26,
1:40, DSHB) (see Notes 10–12).
11. Remove the primary antibodies, and wash the sample using
PBT for 5 min. Repeat this step for another three times.
12. Add secondary antibodies diluted in 3 % BSA and allow incu-
bation for 2–4 h in the dark (see Notes 13–15).
13. Remove the secondary antibodies, and wash the sample with
PBT three times. Each wash should last for at least 5 min.
14. Remove PBT and incubate the ovaries with DNA-staining dye,
such as TO-PRO-3 Iodide (1:5,000–1:10,000, Invitrogen) or
Hoechst (1:5,000–1:10,000, Invitrogen), dissolved in PBT,
for 20–30 min.
15. Remove the dye completely and add one drop of mounting
medium (Vector Laboratories, Inc.) into the tube such that the
ovaries are completely submerged. The fluorescence can be
stably maintained at −20 °C for 2 weeks.
3.3 Mounting and 16. Cut the end of a yellow pipette tip and use it to transfer the
Result Presentation stained ovaries onto a clean glass slide with minimal volume of
mounting medium (see Note 16).
Fig. 3 The process of removing muscle sheath. (a) The anterior structure of the germarium (yellow arrow) is
hidden from view before removal of the muscle sheath (white arrow). (b) After removing the muscle sheath
(white arrow), the niche structure is revealed. Yellow arrow indicates the stack of TF cells
Fig. 4 Examples of fluorescence-labeled adult ovaries. The nuclei are stained by To-pro3 (Invitrogen). (a) Anti-
LamC (red, DSHB) strongly labels TF cells and CCs (white arrowheads). Anti-α-Spectrin (red, DSHB) marks
spectrosome in GSCs and cystblast (white arrows) and fusome in differentiated germ cells (yellow arrows).
Anti-Vasa (green) labels germline cells. ECs are pointed out by yellow arrowheads. (b) Anti-LamC (green) out-
lines TF cells and CCs (white arrowheads); anti-α-Spectrin (green) labels spectrosome in GSCs and cystblast
(white arrow) and fusome in differentiated germ cells (yellow arrows). Anti-pSMAD1-5 (red, Cell Signaling)
labels GSCs. Scale bar: 10 μm
17. Place a needle at the posterior part of the ovary to hold it. Use
another needle to dissociate it into individual ovariole.
18. Place one needle onto a late-stage egg chamber to hold the
ovariole stably while using another needle to peel off the mus-
cle sheath by sliding the needle along the slit between the mus-
cle sheath and the ovariole (Fig. 3) (see Note 17).
19. Place a cover glass directly over the sample. Now the sample is
ready to be examined under the fluorescence microscope
(Fig. 4).
4 Notes
1. 20 % PFA is stable at 4 °C for 6 weeks, after which its fixing

efficiency will diminish due to its gradual degradation.
2. The fixative buffer should be freshly prepared.
6 Lichao Luo et al.
3. Other fixative buffers such as 4 % formaldehyde solution and

methanol may also be used. We recommended PFA here for its
consistency and robustness.
4. This is to ensure that the flies are provided with sufficient
nutrient to undergo optimum reproduction and to allow the
ovaries to assume a healthy structure. For best result, the flies
should not be chosen from an overcrowded vial as they may be
nutritionally deprived. The optimum number of flies to hatch
from a vial (containing approximately 8 ml of food) should be
kept below 50.
5. Any equipment which provides gentle mixing/rocking can be
used: lab quake, nutating mixer, shaker, and rotator or manu-
ally by hands.
6. For dissection of young adult flies (within 24 h post eclosure)
that have smaller ovaries, it is advisable to retain the body part
directly connected to the ovaries, such as the skin at the end of
the abdomen. This is to assist visualizing and locating the ova-
ries during immunostaining.
7. If the ovaries tend to adhere to the wall of the microfuge tube,
coat the tube with 3 % BSA buffer at room temperature for at
least 30 min with rotation. Coating is not necessary if the users
do not encounter such a problem.
8. For staining of phosphorylated proteins such as pMad or pErk,
increasing the fixing time to 40 min and adding phosphatase
inhibitor (1:200, Sigma) to the fixative buffer may help to
improve staining quality.
9. When fixing sample on a nutating mixer, ensure that the ova-
ries are always submerged in the buffer/solution and do not
become clumpy.
10. Anti-Lamin C strongly outlines the nuclear membrane of TF
cells and CCs. Anti-α-Spectrin stains the spectrosomes of GSCs
and cystoblasts as rounded or bar shape (dividing cells) struc-
ture, and the fusomes of differentiated cysts as branched struc-
ture [14] (Fig. 4).
11. Other antibodies can also be used to label GSCs and the niche,
such as anti-Hts (DSHB). It is recommended to find out the
optimum dilution factor of these antibodies prior to the
experiments.
12. For multiple staining with other antibodies (see Fig. 4), add
this antibody after step 11 and incubate it for 2–4 h, followed
by washing with PBT three times and 5 min each time.
13. The staining can also be done at 4°C overnight.
14. The samples should always be kept in the dark when incubat-
ing with secondary antibodies to avoid losing the fluores-
cence signal.
15. We recommend using cy3-conjugated antibody (Jackson

ImmunoResearch LABORARORIES, INC.) at 3 μg/ml, and
other fluorescent-conjugated antibodies (Jackson
ImmunoResearch LABORARORIES, INC.) at 1–3 μg/ml.
16. The recommended volume of mounting medium per slide is
about 30 μl (for cover glass measuring 22 mm × 32 mm).
Excessive medium may cause movement of the ovariole and
affect observation under higher magnification lenses.
17. The slit can be easily seen at the boundary of two chambers
(Fig. 3a white arrow).
Acknowledgments
This work was supported by Temasek Life Sciences Laboratory

(TLL) and Singapore Millennium Foundation.
References
1. Kirilly D, Xie T (2007) The Drosophila ovary: nizes encapsulation by somatic support cells.
an active stem cell community. Cell Res Development 129:4523–4534
17:15–25 9. Liu M, Lim TM, Cai Y (2010) The Drosophila
2. Lin H (2002) The stem-cell niche theory: les- female germline stem cell lineage acts to spa-
sons from flies. Nat Rev Genet 3:931–940 tially restrict DPP function within the niche.
3. Losick VP, Morris LX, Fox DT, Spradling A Sci Signal 3:ra57
(2011) Drosophila stem cell niches: a decade 10. Eliazer S, Shalaby NA, Buszczak M (2011)
of discovery suggests a unified view of stem Loss of lysine-specific demethylase 1 nonauto-
cell regulation. Dev Cell 21:159–171 nomously causes stem cell tumors in the
4. Xie T, Spradling AC (2000) A niche maintain- Drosophila ovary. Proc Natl Acad Sci USA
ing germ line stem cells in the Drosophila 108:7064–7069
ovary. Science 290:328–330 11. Streit P, Reubi JC (1977) A new and sensitive
5. Xie T, Spradling AC (1998) Decapentaplegic staining method for axonally transported
is essential for the maintenance and division of horseradish peroxidase (HRP) in the pigeon
germline stem cells in the Drosophila ovary. visual system. Brain Res 126:530–537
Cell 94:251–260 12. Goetsch JB, Reynolds PM, Bunting H
6. Song X, Wong MD, Kawase E, Xi R, Ding BC, (1952) Modification of Gomori method for
McCarthy JJ, Xie T (2004) Bmp signals from alkaline and acid phosphatase avoiding arti-
niche cells directly repress transcription of a fact staining of nucleus. Proc Soc Exp Biol
differentiation-promoting gene, bag of mar- Med 80:71–75
bles, in germline stem cells in the Drosophila 13. Watanabe S, Kimura Y, Honda M, Sasaki J
ovary. Development 131:1353–1364 (1994) Selective staining of the superficial cells
7. Song X, Xie T (2002) DE-cadherin-mediated of mouse urinary bladder epithelium by
cell adhesion is essential for maintaining horseradish peroxidase (HRP). J Electron
somatic stem cells in the Drosophila ovary. Microsc (Tokyo) 43:119–121
Proc Natl Acad Sci USA 99:14813–14818 14. de Cuevas M, Spradling AC (1998)
8. Schulz C, Wood CG, Jones DL, Tazuke SI, Morphogenesis of the Drosophila fusome and
Fuller MT (2002) Signaling from germ cells its implications for oocyte specification.
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Chapter 2
Genetic, Immunofluorescence Labeling, and In Situ

Hybridization Techniques in Identification of Stem Cells
in Male and Female Germline Niches
Shree Ram Singh, Ying Liu, Madhuri Kango-Singh, and Eviatar Nevo
Abstract
Stem cells have an enormous capacity of self-renewal, as well as the ability to differentiate into specialized
cell types. Proper control of these two properties of stem cells is crucial for animal development, growth
control, and reproduction. Germline stem cells (GSCs) are a self-renewing population of germ cells, which
generate haploid gametes (sperms or oocyte) that transmit genetic information from generation to genera-
tion. In Drosophila testis and ovary, GSCs are anchored around the niche cells. The cap cells cluster in
females and hub cells in males act as a niche to control GSC behavior. With highly sophisticated genetic
techniques in Drosophila, tremendous progress has been made in understanding the interactions between
stem cells and niches at cellular and molecular levels. Here, we provide details of genetic, immunofluores-
cence labeling, and in situ hybridization techniques in identification and characterization of stem cells in
Drosophila male and female germline niches.
Key words Drosophila, Testis, Ovary, Germline stem cells, Niches
1 Introduction
In recent years, the stem cell field has opened a new venue in
regenerative and reproductive medicine. Stem cells have an enor-
mous ability to self-renew as well as produce diverse types of dif-
ferentiated cells [1–4]. Stem cells provide an opportunity to dissect
the cellular and molecular mechanisms controlling embryonic
development, cellular differentiation, and organ maintenance and
also have great potential in developing novel cell-based therapies.
In order for stem cells to function properly, a tight balance between
proliferation and differentiation should be maintained because
over-proliferation of stem cells results in tumor formation [5],
while under-proliferation results in loss of stem cell population,
which results in an inability to form the specific tissue or organ
[1, 6]. A large body of research suggests that stem cells are regu-
lated by specific microenvironments, known as niches, which is a
9
10 Shree Ram Singh et al.
subset of neighboring stromal cells and extracellular substrates.

The stromal cells usually secrete growth factors to regulate stem
cell function [1].
Germline stem cells (GSCs) serve as a reservoir for the con-
tinuous production of gametes in all organisms. The existence of
stem cells in the germline was proposed over a century ago [7].
Recent studies in C. elegans, Drosophila, and mouse have provided
detailed molecular mechanisms, which regulate GSC division and
maintenance. GSCs are known to exist in the testes and ovaries of
all animal species [1, 6, 8–10]. Although the niche hypothesis was
first postulated for hematopoietic stem cells, the GSC niches in
Drosophila are the best studied because of well-defined structures
and availability of molecular markers [2, 6, 9]. Using Drosophila as
a model organism, tremendous progress has been made in under-
standing molecular mechanisms underlying interactions between
stem cells and niches. GSCs are present in the gonads of Drosophila
females and males. The proper maintenance and correct differen-
tiation of GSCs are essential for fertility and fecundity.
The Drosophila testis provides an excellent in vivo system to
study stem cells’ niche interactions at the cellular and molecular
levels [3, 11–33]. In Drosophila males, the stem cell niche and the
germline and somatic stem cells (also known as cyst progenitor
cells (CPCs)) are located at the closed anterior apex of each testis.
Each testis has 5–9 GSCs that are encysted by two CPCs. Both
GSCs and CPCs are physically attached to a group of 12 nondivid-
ing somatic cells called the hub [6, 11–15] (Fig. 1), a niche struc-
ture, which supports the self-renewal of GSCs and CPCs [6]. Each
GSC divides in an asymmetric way with the mitotic spindle orien-
tated perpendicular to the hub [12–14]. One of the daughter cells
remains in contact with the hub, inherits the mother centriole, and
retains GSC identity, while the other daughter cell, called a gonial-
blast (GB), inherits the daughter centriole and initiates differentia-
tion (Fig. 1) [12–14]. Similarly, CPCs self-renew and produce
daughters, which differentiate into somatic cyst cells (SCC) [18,
19]. CPCs also produce hub cells [20]. GSCs and gonialblasts con-
tain a spectrosome. The gonialblast will undergo four rounds of
mitotic division with incomplete cytokinesis to form 16 intercon-
nected spermatogonia, which contain a branched fusome. SCC
will grow without further division; but they become elongated,
and form a thin layer around the spermatogonial cyst [21–27].
However, the germ cells form spermatocytes that will increase in
size and ultimately undergo meiosis and differentiate into sperm
[27]. The adhesion between niche cells and stem cells controls self-
renewal in the Drosophila testis germline [6, 11–33]. In addition,
there are several signaling pathways known to control the behavior
of GSCs/CPCs and their niches in Drosophila testes [11–42].
The adult Drosophila contains two ovaries and each ovary is
composed of approximately 16–20 tubes called ovarioles, each
with a specialized structure called germarium. The anterior tip of
Genetic, Immunofluorescence Labeling, and In Situ Hybridization Techniques… 11
a Spermatogonia b c DAPI
Cyst cells
GSCs
CPCs
Hub
cells
VASA
Sox100B
Gonialblast
Stage 15
d Arm 1B1 e FasIII f Zfh-1

BamC DAPI
VASA DAPI
*
b-gal Arm g h i
1B1 Dapi *
Fig. 1 Immunostaining and in situ hybridization of Drosophila testes. (a) Schematic diagram highlights the tip
of the testes, which usually contain five to nine GSCs; only two GSCs are shown in this diagram, surrounded
by about twice as many cyst progenitor cells (CPCs). Both GSCs and CPCs anchor around the hub cells, which
act as niche cells. The testis proliferation center consists of hub, GSCs, CPCs, gonialblasts, and 2- to 16-cell
spermatogonia. (b) Wild-type stage 15 embryonic testes stained with anti-Vasa (red ) to mark the germline and
anti-Sox100B (green) representing the male- specific somatic gonadal precursor (msSGP) cells. (c) Wild-type
testes stained with DAPI. (d) Wild-type testis stained with anti-Arm (green) to mark the hub cells, 1B1 to mark
the spectrosomes and fusome (green), and anti-Vasa (red) marks all germ cells including GSCs. (e) Wild-type
testis stained with anti-BamC to mark the spematogonial cells (white color positive cells inside black dotted
lines) and anti-FasII to mark hub cells (black arrow ). (f) Wild-type testis stain with anti-Zfh-1 (red) to mark cyst
stem cells. (g) Wild type 6 days after heat shock GSC clones highlighted by dotted lines. Testis stained with
anti-β-galactosidase (red ), anti-Arm and anti1b1 (green). (h) Wild-type testis with Gef26 mRNA expression.
(j) Wild-type testis with upd mRNA expression (red ) using Fast red FISH. Dapi marks DNA (blue) in (d, f, g).
Scale bars: 20 µm (b); 10 µm (d-G, I)
a DAPI b SSC cyst FC

ESCs
GSC
CC
TF
IGS CB
c Arm 1b1 VASA DAPI d hh-GFP e FasIII

BamC
f g h
FSC
FC
GFP
DE-Cad DAPI
VASA DAPI
Fig. 2 Immunostaining and in situ hybridization of Drosophila ovary. (a) Wild-type ovary stained with DAPI.
(b) Schematic diagram highlights the tip of the ovary, which contains 2–3 GSCs and types of cells, depicted in
the figure. (c) Wild-type ovary stained with anti-Arm (green) to mark the cap cells, anti-1B1 (green) to mark the
fusome and spectrosomes (green), as well as niche cells and anti-Vasa (red) mark all germ cells including
GSCs. (d) hh-Gal4 UAS-GFP line stained with anti-GFP (green) marks the terminal filament cells and cap cells.
(e) Wild-type ovary stained with anti-Decad (green) and anti-Vasa (red). (f) Wild-type ovary stained with anti-
BamC to and anti-FasIII. (g) ) Wild-type ovary with Gef26 mRNA expression. (h) Wild type 6 days after heat
shock somatic stem cells clones (GFP-green), Dapi marks DNA (red). Dapi marks DNA (blue) in (c, d, f). Scale
bars: 50 µm (c); 20 µm (d); 10 µm (e-h)
each germarium contains three types of stem cells: GSCs, escort

stem cells (ESCs), and follicle stem cells (FSCs). The female GSC
niche contains 5–7 nondividing somatic cap cells, which physically
anchor 2–3 GSCs in each germarium [43–45]. There are 8–10
terminal filament (TF) cells anterior to cap cells connecting the
germarium with the inner germarium sheath (IGS) cells (Fig. 2).
Through asymmetric division, GSC produces self-renewing GSC,

and a differentiating daughter cell called cystoblast (CB), which
moves away from niche and forms an interconnected 16-cell cyst
by incomplete cytokinesis. The cystoblasts move away from the
niche wrapped by differentiated escort cells produced from ESCs.
The cyst cells are encysted by the escort cells until they reach
the 16-cell stage. Only 1 out of 16 germ cells can become an
oocyte and the remaining cells will then become nurse cells to
support the growth of the oocyte. Studies have shown that cap
cells and ESCs interact together to form the GSC niche [43].
Furthermore, it has been shown that DE cadherin is required for
anchoring GSCs in their niche [44]. Several signaling pathways are
responsible for niche stem cells’ interaction and maintenance in the
Drosophila ovary [29–45]. There are 4–6 ESCs and their progeny
are called escort cells [42]. Each germarium contains 40–50 IGS
cells [26] and about 18 escort cells [42]. In addition, there are 2–3
FSCs located in the middle of each germarium across from each
other. FSCs divide and produce a population of mitotically active
follicle progenitor cells, which proliferate in the egg chambers of
stage 1 to stage 6, and produce several types of differentiated cells
that cover egg chambers including a follicle cell monolayer [43].
IGS cells and cap cells act as an FSC niche and FSC behavior is
regulated by several signaling pathways [26, 27, 44]. In this chap-
ter, we provide the protocols (immunostaining, generation of
germline and somatic clones, and in situ hybridization, see Figs. 1
and 2) to identify and characterize germline and somatic stem cells
in the Drosophila testis and ovary.
2 Materials
Prepare and store all reagents at room temperature (unless other-

wise indicated).
2.1 Culturing 1. Control and transgenic Drosophila lines.

Drosophila 2. Drosophila food: 175 g Brewer’s yeast, 525 g corn meal, 103 g
sugar, 75 g agar, 17.5 g baker’s yeast, and methyl paraben
solution (add 26.6 g methyl paraben in 15.4 propionic acid
and 105 ml ethanol—dissolve by heating and stir). To prepare
the fly food add 7.91 l of double-distilled water to the above
gradients and mix well, autoclave for 45 min, take out, and mix
thoroughly. When cooked food temperature is 85 °C add the
methyl paraben solution and mix well.
3. Autoclave for food preparation.
4. Fly food dispenser.
5. Fly plastic vials.
6. Plastic bottles.
7. Foam or cotton plugs.

8. Morgue containing 70 % alcohol for discarding the dead flies.
9. Fly trap to make sure that flies escaped during crossing have
been trapped.
10. Fly culture incubators (18, 25, and 29 °C).
2.2 Isolation 1. 3–5-day-old male and female flies of control (Oregon-R) and
of Testis and Ovary transgenic lines.
2. Dissecting solution (Drosophila Ringer’s solution): 130 mM
NaCl, 4.7 mM KCl, 1.9 mM CaCl2, and 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
pH 6.9. Dissolve 7.5 g NaCl, 0.35 g KCl, 0.21 g CaCl2, and
2.38 g HEPES in approximately 1 l distilled water and stir to
dissolve. Adjust to pH 7.2 with 1 N HCl and make the final
volume of 1 l with distilled water. Store the dissecting solution
in a glass bottle at 4 °C or it can be stored at room temperature
for a short time.
3. Drosophila anesthesia CO2 station.
4. Drosophila CO2 fly pads.
5. Paint brush.
6. Dissecting tweezers.
7. Glass microslides.
8. Plastic dropper.
9. Kimwipes.
10. Dissecting microscope.
11. Ice.
12. 70 % (v/v) ethanol.
13. Pipet (20, 200, 1,000 μl).
14. Pipet tips (200, 1,000 μl).
2.3 Generation 1. Stocks for generating FLP-mediated recombination germline

of Germline and clones: hsFLP; FRT 82B arm-lacZ; FRT 40A arm-lacZ. These
Somatic Clones stocks are available from the Bloomington Stock Center
(http://www.flybase.org).
2. Stocks for generating CPC clones using MARCM system:
c587-Gal4.UAS-2XEYFP/FM7; FRT 40A-tub-Gal80/Cyo;
40A +
FRT -w /Cyo; +/TM3, Sb, hs-Flp. For details, see ref. 33.
3. 37 °C water bath tank for heat-shock regime.
4. 25 °C incubator to maintain fly crosses.
5. Antibodies: Mouse or rabbit anti-GFP and mouse or rabbit
anti-β-galactosidase.
2.4 Immunostaining 1. Phosphate-buffered saline (PBS): 130 mM NaCl, 7 mM

and Microscopy Na2HPO4, 3 mM NaH2PO4, and adjust to pH 7.4 with HCl.
of Testis and Ovary Store at room temperature. For longer stability of the solution,
store at 4 °C.
2. Triton X-100 or Tween-20 (Sigma).
3. PBX solution: 0.1 % Triton X-100.
4. Fixation solution: 4 % (w/v) paraformaldehyde in 1× PBX.
5. Gloves.
6. Parafilm.
7. Eppendorf tubes.
8. 15-ml conical tubes.
9. 1.5-ml microcentrifuge tubes.
10. Centrifuge.
11. Blocking solution: 2 % normal goat serum (Vector laborato-
ries) in 1× PBX. Store at 4 °C.
12. Bovine serum albumin (BSA; Sigma).
13. Minivortex (VWR Scientific Products).
14. Tube shaker.
15. Aluminum foil.
16. Microcentrifuge tube rack (Fisher Scientific).
17. Primary antibodies: Rabbit anti-Vasa (1:2,000), Mouse anti-
1b1 (1: 20), Rat anti-Decad (DSHB, 1:20), Mouse anti-FasIII
(DSHB, 1:100), Mouse anti-Arm (DSHB, 1:200), Mouse
anti-BamC (DSHB, 1:10), Guinea pig anti-Zfh-1 (1:4000),
Rat anti-Tj (1:400), Rabbit anti-Sox 100B (1:1,000), Rabbit
or Mouse anti-β-galactosidase (Invitrogen, 1:500), and Rabbit
or Mouse anti-GFP (Invitrogen, 1:500) for GFP-fusion pro-
tein lines. The above antibodies can be stored at 4 °C for short
term. For long-term storage, use −20 °C with 50 % glycerol or
−80 °C. For details about the antibodies used in GSCs, see
refs. [33–61].
18. Secondary antibodies: Goat anti-Mouse, Goat anti-Guinea
pig, Goat anti-Rat, and Goat anti-Rabbit, conjugated to Alexa
Fluor 488 or Alexa Fluor 594 or Texas Red (Invitrogen). Store
in the dark at 4 °C. Use 1:200–500 dilutions in 1× PBX.
19. DAPI (4,6-Diamidino-2-phenyldole dihydrochloride)
(Invitrogen) to stain DNA. Store in the dark at 4 °C.
20. 50 % glycerol in 1× PBS.
21. 4, –20, and −80 °C freezers.
22. Permanent marker.
23. Microscope cover glass.
24. Microscope slides.
25. Light, fluorescent, and confocal microscope.

26. Microslide plastic folder.
27. Computer and software for image processing.
2.5 Collection, 1. Drosophila adult flies.

Preparation, Fixation, 2. Grape juice.
and Staining
3. Culture plate.
of Embryos
4. Washing buffer (10×): 70 g NaCl and 3 ml Triton X-100, dis-
solve in 1 l of water.
5. Nylon mesh.
6. 50 % Clorox bleach.
7. Triton X-100.
8. Paintbrush.
9. Eppendorf tubes.
10. Glass scintillation vial.
11. 5× PEM: 0.5 M Pipes, pH 6.9, 10 mM MgSo4, 5 mM EGTA,
pH 7.0.
12. Fixation solution: 1.375 ml dH2O, 0.5 ml 5× PEM, 0.625 ml
16 % formaldehyde, 2.5 ml heptane.
13. Plate form shaker.
14. Pasteur pipet.
15. Heptane.
16. Methanol.
17. 2 % Normal goat serum.
18. 1× PBX.
19. Primary antibodies. See Subheading 2.4.
20. Secondary antibodies. See Subheading 2.4.
21. 50 % glycerol.
2.6 In Situ 1. Dissecting solution (Drosophila Ringer’s solution): See

Hybridization Subheading 2.2.
2. 4 % paraformaldehyde (Sigma).
3. HEPES buffer: 0.1 M HEPES pH 6.9, 2 mM MsSo4, 1 mM
ethylene glycol tetraacetic acid (EGTA).
4. PBT: 1× PBS, 0.1 % Tween-20.
5. Proteinase K (Sigma).
6. Glycine.
7. Water bath.
8. Moist chamber.
9. Ice.
10. 96-well plate.

11. RNAse-free water.
12. Hybridization buffer: 50 % deionized formamide, 5× SSC,
100 μg/ml sonicated salmon sperm DNA, 50 μg/ml heparin,
0.1 % Tween-20.
13. N-methylthiotetrazole (NMTT): 100 mM NaCl, 50 mM
mgCl2, 100 mM Tris pH 9.5, 0.1 % Tween-20.
14. Alkaline phosphatase conjugated anti-DIG antibody
(Boehringer Mannheim).
15. NBT (Nitro-Blue Tetrazolium) (Boehringer Mannheim).
16. X-phosphate (Boehringer Mannheim).
17. Fast Red tablets (Santa Cruse). To use the tablet for staining,
dissolve one Fast Red tablet in 2 ml 0.1 M Tris–HCl, pH 8.2.
Filter using 0.2 μm filter. Shake for 1–3 min. Use the prepared
solution within 30 min after preparation. Tablet should be
stored at −20 °C (see Note 1).
18. Glycerol.
19. DAPI solution.
3 Methods
3.1 Generation 1. Clones of mutant GSCs were generated by Flp-mediated

of Germline Clones mitotic recombination, as described previously [43].
2. To generate the stocks for GSC clonal analysis, produce the
flies carrying an armadillo-lacZ transgene in trans to the
mutant-bearing chromosome using standard crosses [30].
3. Take the 3–5-day-old adult males or females carrying an arm-
lacZ transgene in trans to the mutant-bearing chromosome
and heat-shock them for 1 h at 37 °C for 3 consecutive days,
separated by 8–12 h of interval in each heat shock (see Note 2).
4. After the heat shock, transfer the males to fresh food every day
at room temperature.
5. Remove the testis and ovary after 2 days, 4 days, 6 days, and 2
weeks after the last heat-shock treatment for antibody staining.
3.2 Generation 1. CPC clones can be generated using the MARCM system [61].
of Somatic Clones 2. Cross the c587-Gal4.UAS-2XEYFP/FM7; FRT40A-tub-Gal80/Cyo
virgin females with males of FRT40A-w+/Cyo; +/TM3, Sb,
hs-Flp.
3. Heat-shock 3–5-day-old males and females carrying a tub-
Gal80 transgene in trans to the mutant-bearing chromosome
for 1 h at 37 °C for 2 days separated by 8–12 h of interval on
each heat shock.
4. Transfer the males and females at 25 °C to fresh food vial every

day.
5. Remove the testis and ovary 2, 4, 6 days and 2 weeks after the
final heat-shock regime for antibody staining.
3.3 Isolation 1. Collect adult males and females after 3–5 days of emergence
of Testis and Ovary under CO2 station.
2. Place a slide under dissecting microscope and put drops of dis-
secting solution on the slide.
3. Take the males and females using tweezers. Put one tweezers
at the thorax region and with other tweezers take out the ter-
minalia and isolate the ovaries and testes.
4. Transfer the dissected testis and ovary into separate tubes con-
taining dissecting solution in ice.
5. For ovary, it is better to dissociate ovaries into ovarioles and
then fragment each ovariole into pieces with fine tungsten nee-
dles before putting in dissecting solution or fixation solution.
3.4 Immunofluore- 1. Fix the tissues in 4 % formaldehyde for 30 min.

scence Staining of 2. Remove the fixative solution and wash the testes and ovaries
Testis and Ovary three times for 2 min each in 1× PBX (see Note 3).
3. Block the tissues in 2 % normal goat serum for overnight at
4 °C or 30 min at room temperature.
4. Prepare the primary antibodies in specific concentration in
1× PBX. Incubate the tissues with primary antibodies over-
night at 4 °C.
5. Then wash the tissues at room temperature for 15 min in 1×
PBX three times.
6. Dilute the secondary antibodies in desired concentration and
incubate the tissue for 2 h at room temperature (see Note 4).
7. Remove the secondary antibodies and wash the tissue for 15
min in 1× PBX three times.
8. After the final wash, counterstain the tissues in DAPI for 5 min.
9. Rinse the tissues with 1× PBS, put 50 % glycerol as a mounting
medium in the tubes, and put the tubes in 4 °C.
10. Next day, using the dissecting microscope, put the tissue on a
glass slide, arrange the testis and ovary in the desired direction,
and cover with glass slides.
11. Image the tissues using confocal microscopy.
12. The number of GSCs and somatic stem cells can be determined
using serial confocal reconstructions of the entire testis and
ovary tip. Details of the specific markers expressed in each cell
type in the testis and ovary are presented using the above pro-
tocol in Figs. 1 and 2.
13. Using the above protocols, many diverse types of antibodies can
be tested to study male and female GSCs. For references and
details of the antibodies, see refs. [9–60].
3.5 Collection, 1. Collect embryos on grape juice plates overnight.

Preparation, Fixation, 2. Rinse embryos in washing buffer into a nylon mesh.
and Staining
3. Dechorionate by putting embryos in 50 % Clorox for 5 min at
of Embryos
room temperature.
4. Rinse embryos twice with washing buffer.
5. Transfer the embryos to fixation solution containing heptanes
in a glass scintillation vial, put the vials on shaker, and shake at
a moderate speed for 25 min.
6. Discard the aqueous (bottom) layer of solution with the help
of a Pasteur pipet.
7. Transfer the embryos in 10 ml of heptane.
8. Add 10 ml of methanol to the embryos and shake vigorously
for 30–60 s to help in devitellinization of embryos.
9. Transfer the devitellinized embryos to a fresh tube and rinse
with methanol.
10. Embryos can be stored at −20 °C for extended period for
future staining.
11. To stain the embryos, take a desired number of embryos in
Eppendorf tubes.
12. Rehydrate the embryos into the following order: 9:1 metha-
nol:1× PBX; then put 7:3 methanol:1× PBX, then put 5:5
methanol:1× PBX; then put 3:7 methanol:1× PBX, then put
1:9 methanol:1× PBX, and then wash with PBX two times.
13. Remove PBX and incubate the embryos in 2 % normal goat
serum.
14. The primary and secondary antibody dilution, incubation,
washing, and microscopy can be done in a similar way as men-
tioned in Subheading 3.4.
3.6 In Situ 1. Probes can be prepared using SP6/T7DIG RNA labeling kit
Hybridization (Roche) following the manufacturer’s instructions.
(See Note 5) 2. Dissect the 3–5-day-old males and females as mentioned in
Subheading 3.3.
3. Fix the tissues for 30 min in 4 % paraformaldehyde in HEPES
buffer at room temperature.
4. Wash the tissues three times for 5 min each in 1× PBX.
5. Incubate the tissues in 50 μg/ml proteinase K for 5 min
(see Note 6). Stop the reaction with 2 mg/ml glycine for 2 min
and wash the tissues two times for 5 min each with 1× PPX.
6. Fix and wash the tissues again if using step 5. If not, wash the
tissues for 10 min in 1:1 ratio of 1× PBX:hybridization
buffer.
7. Pre-hybridize the tissues for about 1 h at 65 °C in a water bath
(for RNA probes) in hybridization buffer. It is advised to pre-
heat the hybridization buffer.
8. Denature the probe by heating at 70 °C for 10 min in a water
bath, and then rapidly cooling in ice.
9. Then heat the hybridization buffer at 65 °C in a water bath,
mix the probe (in a ratio 1:50 probe:hybridization buffer), and
put on tissue.
10. Hybridize overnight at 65 °C in a water bath. Make sure to
have moist chamber where you can place the tube. No need to
shake.
11. Next day, after hybridization, wash the tissues six times for
30 min each in hybridization buffer in a water bath at
65 °C.
12. Then wash for 15 min in 4:1 ratio hybridization buffer:1× PBX
16. Then wash two times for 15 min each in 1× PBX at room
temperature.
17. For nonfluorescent staining with NBT and X-phosphate, incu-
bate the tissue overnight in a 1:2,000 dilution of alkaline
phosphatase-conjugated anti-DIG antibody in 1× PBX at 4 °C;
then three times, 15 min each, with 1× PBX; and after that,
three times for 15 min each with NMTT. Prepare the color
reaction by adding 4.5 μl NBT and 3.5 μl X-phosphate in 1 ml
NMTT. Incubate the tissue for 10–30 min depending on the
color reaction; once you see the color changing in the tissue by
looking at the dissecting microscope, it is better to stop the
reaction by putting 1× PBX. Put in 50 % glycerol for 1 h and
tissue can be put in 90 % glycerol overnight. It is better to
mount in 90 % glycerol. Image can be taken using the micro-
scope with bright-field and DIC capabilities.
18. For fluorescent in situ hybridization (FISH), we used Fast
Red staining procedure by following the manufacturer’s
instructions. Follow the above method from steps 1 to 17,
and then incubate tissues in 2 % blocking solution. Add pri-
mary antibody anti-DIG, incubate the tissues at 37 °C for
1 h, wash, and in the place of secondary antibody use Fast

Red solution. Incubate the tissues with 1 mg/ml of Fast
Red solution and monitor the reaction to prevent over-
staining. Stop the reaction by washing the tissues in water or
1× PBS. Put the 50 % glycerol for mounting. Images can be
taken using a confocal microscope (some of the images
using the above procedures are provided in Figs. 1 and 2).
With the above procedure, two-color and three-color FISH
can also be performed.
4 Notes
1. The Fast Red working solution should be filtered to remove

non-dissolved substrate particles.
2. 8–12 h of interval in each heat shock was used to allow flies to
recover from heat shock because shorter interval can kill the
flies.
3. Proper care should be taken while staining the ovary because
during washing ovarioles can be washed out.
4. Wrap the tubes with foil during secondary antibody staining to
avoid exposure of light and fading of the signal.
5. All the materials should be RNase free; always use gloves and
use DEPC-treated water.
6. Since Drosophila tissues are soft, it is not necessary to use pro-
teinase K.
Acknowledgments
M.K.S. is supported by the Knight’s Templar Eye Foundation and

start-up support from the University of Dayton, OH. We thank
Robin Permut for editing the manuscript.
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Chapter 3
Visualization of Adult Stem Cells Within Their Niches Using

the Drosophila Germline as a Model System
Annekatrin König and Halyna R. Shcherbata
Abstract
The germaria of the fruit fly Drosophila melanogaster present an excellent model to study germline stem
cell–niche interactions. Two to three adult stem cells are surrounded by a number of somatic cells that
form the niche. Here we describe how Drosophilae germaria can be dissected and specifically immuno-
stained to allow for identification and analysis of both the adult stem cells and their somatic niche cells.
Key words Drosophila, Germarium, Ovary, Adult stem cells, Stem cell niche, Germline, Ovarian
soma, Immunostaining
1 Introduction
Adult stem cells usually reside in the stem cell niche, a unique
physiological microenvironment that helps stem cells to carry on
self-renewing divisions throughout the lifetime of an organism.
The niche includes cellular and noncellular elements that can be
divided into one of the two main mechanistic types—physical con-
tacts and diffusible factors [1]. Close contacts include tight junc-
tions, adherens junctions, gap junctions, the Notch signaling
pathway, the basement membrane, and extracellular matrix pro-
teins. Diffusible factors, which are secreted by niche cells and travel
over varying distances to keep stem cell identity, often affect tran-
scription. Stem cells must be anchored to the niche through cell–
cell interactions so that they will stay both close to niche factors
that specify self-renewal and far from differentiation stimuli.
Presently the existence of a stem cell niche has been demon-
strated for mammalian adult stem cells in the hematopoietic, epi-
dermal, neural, and intestinal systems. However, the stem cell
niches involved in maintenance of adult mammalian tissues and
particularly their role in cancer development remain complex,
poorly defined, and difficult to study in vivo [2].
25
26 Annekatrin König and Halyna R. Shcherbata
Fig. 1 Scheme of larval ovary and adult germarium. (a) The primordial germ cells (PGC) that can be identified
by their characteristic spherical spectrosomes (SS spherical skeletal organelles) are intermingled with somatic
cells (IC, intermingled cells). Stacks of the terminal filament (TF) cells have already formed in late L3 larvae.
Cap cells (CpCs) are forming in late L3 larvae through the early pupal stages at the base of TFs. Two popula-
tions of somatic cells (APC, apical cells, BC, basal cells) are also found in the larval ovary. (b) In the adult
ovaries, the individual ovarioles with the germaria are separated by peritoneal sheath. The germline stem cells
(GSCs) are positioned at the anterior of the germarium and directly attached to cap cells. Upon asymmetric
division, the stem cells give rise to another stem cell and a differentiating daughter, the cystoblast (CB). The
cystoblast divides four more times with incomplete cytokinesis, forming the cyst. During that process, the
spherical spectrosomes of the GSCs elongate and branch to form the fusome (Fu). The terminal filament cells
are in close proximity to the cap cells, but have a more oval shape. The GSCs are furthermore in contact with
another type of somatic cells that presents an important component of the niche: the escort cells (EC). Follicle
cells (FC) that are produced by follicle stem cells (FSC) encapsulate the developing egg. Anterior is to the left
The Drosophila ovarian stem cell niche is very well characterized

and has been used for many years to unravel the complex stem cell–
niche interactions. The insights gained from these studies led to a
better understanding of how stem cells work: in addition to cell–cell
interactions [3] between stem and niche cells, a variety of signaling
pathways involved in stem cell control were described [4–13].
The easily identifiable and analyzable cells in the Drosophila
germline niches and the sophisticated genetic tools that are available
in Drosophila make it an ideal system for studying stem cell–niche
interactions [14]. The paired ovaries of the adult female fly each
consist of 16–20 ovarioles that contain developing egg chambers.
Located at the anterior of every ovariole is the germarium, where
two to three stem cells are held by 5–7 cap cells and are in contact
with other somatic cells (see Fig. 1b). By asymmetric division, the
adult stem cells give rise to both new stem cells and differentiated
cells that will become the egg. The differentiated germline cells are
surrounded by somatic escort cells that are another important com-
ponent of the stem cell niche [4, 15]. More posteriorly, follicle cells
that are generated by specific stem cells encapsulate the differentiat-
ing germline [16]. The individual ovarioles are held together by the
Visualization of Adult Stem Cells Within Their Niches Using the Drosophila Germline… 27
Fig. 2 Pre-adult ovary and adult germaria. (a) In the late larval and early pupal ovaries, terminal filament stacks
become visible (outlined in yellow ). The PGCs (arrow ), that can be identified by their spherical SSs, are not
separated yet into individual ovarioles and intermingled with somatic cells. (b) Adult ovaries consist of several
germaria, each containing 2–3 GSCs, that can be not only identified by their characteristic Adducin-marked
SSs but also stained with the stem cell marker pMad. Directly attached to the stem cells are several somatic
cells that are forming the stem cell niche: the CpCs can be marked using LaminC or Engrailed. DE-Cadherin
staining shows the adhesion contacts between the GSCs and CpCs. Furthermore, ECs and CpCs are marked
here with Traffic jam
terminal filaments. A variety of different markers makes it possible to

nicely immunostain and analyze number, localization, shape, and
interactions of the individual cells (see Fig. 2b). In addition, the
development of the stem cell niche itself can be directly observed in
developing larvae and early pupae where the cap cells divide and
terminally differentiate (see Figs. 1a and 2b) [17].
In this chapter we show how to visualize adult stem cells in
their niches in adult female Drosophila.
2 Materials
2.1 Fly Husbandry 1. Standard cornmeal agar food (recipes can be found at http://
fly.bio.indiana.edu/).
2. Yeast paste: Dry yeast should be mixed in 5 % propionic acid
(see Note 1).
2.2 Ovary Dissection 1. Ice block for immobilization of the flies.

2. Sharp tweezers.
3. Small Petri dish for dissections.
4. Stereomicroscope for dissection.
5. Pasteur pipettes.
2.3 Fixation 1. Fixing solution: 4 % formaldehyde in phosphate-buffered

and Washing saline (PBS) (see Note 2).
2. Nutator.
3. PBT: 0.2 % Triton X in PBS.
2.4 Antibodies 1. Blocking solution: 0.2 % bovine serum albumin, 5 % normal

goat serum in PBT (see Note 3).
2. Primary antibodies: Many antibodies are available to study
germline–niche interactions; some of these are listed in Table 1.
Dilute primary antibodies in blocking solution and store at
4 °C (see Notes 4–8).
Table 1
A subset of antibodies that are useful to study germline stem cell niche interactions is shown
Name of the Antibody Used to mark in the

Protein recognized antibody Raised in source germarium Dilution
Armadillo N2 7A1 Mouse, DSHB Iowa Cell-Cell adhesion 1:50
IgG2a between cap cells
and between stem
cells and cap cells
Bag of marbles BamC Mouse, Rat D. McKearin Differentiating 1:1,000
(BAM) (cyto- germline cysts, not
plasmic) in germline stem cells
E-Cadherin, 5D3 Mouse, DSHB Cell-Cell adhesion 1:50
extracellular IgG2b Iowa between cap cells
domain and between stem
cells and cap cells
Engrailed 4D9 Mouse, DSHB Cap cells 1:50
IgG1 Iowa
Held out wings HOW Rabbit, T. Volk Germline stem cells, 1:1,000
(HOW) Rat cystoblasts
Hts/Adducin-like 1B1 Mouse, DSHB Spectrosomes 1:50
IgG1 Iowa and fusomes
Lamin C LC28.26 Mouse, IgG1 DSHB Cap cells 1:50
Iowa
Phosphorylated pMad Rabbit E. Laufer Germline stem cells 1:5,000
Mothers against
Dpp (pMAD)
Traffic jam TJ Guinea pig D. Godt Escort cells, cap cells 1:3,000
Vasa Rat P. Lasko Germline cells 1:1,000
Scientists who generated a particular antibody are named as source. Their addresses are available from flybase (http://
flybase.org/). DSHB Developmental Studies Hybridoma Bank at the University of Iowa
3. Secondary antibodies: Conjugated Alexa fluor goat anti-

mouse, goat anti-rabbit, or goat anti-rat from Molecular
Probes, diluted 1:500 in blocking solution (Molecular Probes);
store at 4 °C (see Notes 8 and 9). The secondary antibodies
have to be chosen with respect to the laser lines of the available
microscope.
2.5 DNA Staining 1. DAPI solution: Make a 100× DAPI solution (1 mg/ml) and
and Mounting store aliquots at −20 °C. For staining, dilute in PBS
(see Note 10).
2. Glycerol: 70 % Glycerol, 3 % n-propyl gallate (NPG)
(see Note 11).
3. Tungsten needles.
2.6 Analysis Laser scanning confocal microscope is used for analysis.
3 Methods
3.1 Dissection All steps are carried out at room temperature unless otherwise
stated. During all incubations and washes, the Eppendorf tubes are
placed on a nutator.
3.1.1 Adult Ovaries 1. Immobilize 5–10 female flies by putting them on an ice block.
2. The ovaries are positioned in the abdomen of the fly and are
simple to find in well-fed individuals (see Note 12). Dissect the
flies in 1× PBS using a stereomicroscope, and hold the fly with
one pair of tweezers at the thorax. Carefully open the cuticle at
the posterior end of the animal with another pair of tweezers.
If necessary, gently push the abdomen to squeeze out the
paired ovaries. Remove all remnants of guts and cuticle and
place the ovaries in an Eppendorf tube using Pasteur pipettes
(see Note 13).
3.1.2 Larval Ovaries 1. Pick up late third instar larvae from the wall of the food vial or
bottle.
2. Select a female larva and hold with a pair of tweezers at the
anterior end.
3. The larval ovaries are located in the fat body. Cut off the larval
head and hold the posterior end of the remaining larval body
with one pair of tweezers. Carefully now invert the larvae by
pulling it over the tweezers with another pair of tweezers.
Remove cuticle and guts and transfer the fat body into an
Eppendorf tube or a 24-well plate (see Note 14).
3.1.3 Fixation 1. Add fixing solution and incubate for 10 min. Remove the
fixing solution carefully and wear protective gloves when
handling the fixative.
2. Wash the ovaries three times for 15 min each with PBT
(see Notes 15 and 16).
3.2 Antibody 1. Add blocking solution and incubate for 1 h.

Staining 2. Remove the blocking solution and add primary antibody solu-
tion. Incubate overnight at 4 °C.
3. Remove the antibody solution (see Note 5) and wash the ova-
ries three times for 15 min each with PBT.
4. Block again in blocking solution for 1 h.
5. Incubate the ovaries in secondary antibody solution overnight
at 4 °C or for 3 h at room temperature.
6. Remove the secondary antibody solution and wash with PBT
twice for 15 min. Add DAPI solution and incubate for 10 min.
7. Remove the DAPI solution and wash three times for 15 min
with PBT.
8. Remove as much PBT as possible and add a few drops of glyc-
erol to the ovaries.
(a) Adult ovaries: Place the ovaries on a slide and use tweezers
and tungsten needles to separate the individual ovaries
and to remove the mature eggs.
(b) Larval ovaries: Place the fat bodies on a slide and locate
the larval ovaries. Carefully remove remnants of the fat
body.
9. Place a coverslip on top of the samples and analyze using a
confocal microscope.
4 Notes
1. The yeast paste should have a “peanut butter-like” texture.

The propionic acid helps to avoid fungal or bacterial
contamination.
2. Prepare the solution fresh from a 16 % stock solution at room
temperature.
3. Goat serum is used in the blocking solution if the secondary
antibody was produced in goat. If you have to use secondary
antibodies that were generated in another animal, use other
serums from the appropriate animal.
4. Primary antibodies: Apart from several monoclonal antibodies,
that are available from the Developmental Studies Hybridoma
Bank, a variety of different polyclonal rabbit, goat, sheep, and
guinea pig antibodies have been made by different labs (see

Table 1). However, make sure not to use goat serum in the
blocking solution if the primary antibody is goat derived.
5. When analyzing GFP-marked clonal cells, you may use an anti-
GFP antibody to better visualize the GFP.
6. Stability of primary antibodies: Some primary antibodies can
be reused a couple of times, whereas others can be used only
once. Dilution and stability of every antibody or antibody
batch have to be tested separately.
7. If the antibody staining shows a high level of nonspecific back-
ground, it may be pre-absorbed with fixed embryos: incubate
fixed embryos overnight with the antibody solution. Use this
antibody solution and use it for staining your sample.
8. To avoid bacterial contamination and to extend stability of the
antibody solution 0.05 % of sodium azide can be added.
9. Choose an antibody that targets the animal in which the pri-
mary antibody was produced. Conjugated Alexa fluor antibod-
ies that were raised to target different animals and that have
sufficiently different emission spectra can be combined to
immunostain different antigens at a time. Additionally, if the
primary antibodies are from different antibody subclasses (IgM
or IgG subclasses) secondary antibodies specific to the anti-
body subclass can be used to discriminate the patterns.
Sensitivity and/or cross-reactivity can vary. We have had good
experiences with Alexa 568 goat anti-mouse (emits red light),
combined with for example Alexa 488 goat anti-rabbit/rat
(emits green light) and Alexa 633 goat anti-rat/rabbit (emits
far-red light).
10. If the available confocal microscope does not have a UV laser
illumination system that is necessary to detect DAPI-stained
DNA, you may use propidium iodide to stain the nuclei
instead. Propidium iodide staining: Incubate the ovaries for
15 min in PBS containing 2 μgf/ml propidium iodide. Staining
with DAPI allows you to use three other secondary antibodies
emitting green, red, and far-red light in parallel with DNA
staining, whereas propidium iodide emits red light itself.
11. Add NPG to the glycerol and vortex. If the NPG will not dis-
solve, heat the solution at 37 °C overnight.
12. Oogenesis is highly dependent on the individual’s environ-
ment. Therefore, the flies should be “fattened” on wet yeast
prior to dissection for at least 2 days and should also be kept in
a community with males. However, when analyzing ovarian
phenotypes it is recommended to collect and stain wild-type
and mutant females at different timepoints and in several inde-
pendent experiments.
13. Depending on the antibody used, the immunostaining proto-

col can vary. If the used antibody is also staining the peritoneal
muscle sheath, it is necessary to destroy the sheath around the
ovarioles by sucking them up and down several times in a
Pasteur pipette.
14. The larval fat body that contains the ovaries will not sink to the
ground, but floats in the solution. It is therefore recommended
to check under the stereomicroscope that the fat bodies are
not washed away when adding or removing liquids from the
sample.
15. The ovaries should be fixed as fast as possible after dissection to
preserve the cellular structures. It is important not to exceed
or shorten the fixation time to avoid poor immunostaining.
16. Upon all incubation and washing steps make sure to add an
amount of liquid that is sufficient to allow the ovaries to float
in the tube or the plate upon gentle rocking. Furthermore,
when removing solutions from the tube do not pull up the
ovaries into the pipette and do not damage the ovaries. The
ovaries should stay intact until the very end of the procedure
since the individual germaria are otherwise very easily lost.
References
1. Walker MR, Patel KK, Stappenbeck TS (2009) 7. McKearin D, Ohlstein B (1995) A role for the
The stem cell niche. J Pathol 217(2):169–180. Drosophila bag-of-marbles protein in the dif-
doi:10.1002/path.2474 ferentiation of cystoblasts from germline stem
2. Scadden DT (2006) The stem-cell niche as cells. Development 121(9):2937–2947
an entity of action. Nature 441(7097): 8. Song X, Wong MD, Kawase E, Xi R, Ding BC,
1075–1079. doi:10.1038/nature04957 McCarthy JJ, Xie T (2004) Bmp signals from
3. Song X, Zhu CH, Doan C, Xie T (2002) niche cells directly repress transcription of a
Germline stem cells anchored by adherens differentiation-promoting gene, bag of mar-
junctions in the Drosophila ovary niches. bles, in germline stem cells in the Drosophila
Science 296(5574):1855–1857. doi:10.1126/ ovary. Development 131(6):1353–1364.
science.1069871 doi:10.1242/dev.01026
4. Decotto E, Spradling AC (2005) The Drosophila 9. Konig A, Yatsenko AS, Weiss M, Shcherbata
ovarian and testis stem cell niches: similar somatic HR (2011) Ecdysteroids affect Drosophila
stem cells and signals. Dev Cell 9(4):501–510. ovarian stem cell niche formation and early
doi:10.1016/j.devcel.2005.08.012 germline differentiation. EMBO J 30(8):
5. Xie T, Spradling AC (1998) Decapentaplegic 1549–1562. doi:10.1038/emboj.2011.73
is essential for the maintenance and division of 10. Shcherbata HR, Ward EJ, Fischer KA, Yu JY,
germline stem cells in the Drosophila ovary. Reynolds SH, Chen CH, Xu P, Hay BA,
Cell 94(2):251–260 Ruohola-Baker H (2007) Stage-specific differ-
6. Yu JY, Reynolds SH, Hatfield SD, Shcherbata ences in the requirements for germline stem
HR, Fischer KA, Ward EJ, Long D, Ding Y, cell maintenance in the Drosophila ovary. Cell
Ruohola-Baker H (2009) Dicer-1-dependent Stem Cell. 1(6):698–709. doi:10.1016/j.stem.
Dacapo suppression acts downstream of 2007.11.007
Insulin receptor in regulating cell division of 11. Hatfield SD, Shcherbata HR, Fischer KA,
Drosophila germline stem cells. 2009 Nakahara K, Carthew RW, Ruohola-Baker H
136(9):1497–507. doi:10.1242/dev.025999 (2005) Stem cell division is regulated by the
microRNA pathway. Nature 435(7044): of discovery suggests a unified view of stem

974–978. doi:10.1038/nature03816 cell regulation. Dev Cell 21(1):159–171.
12. Ward EJ, Shcherbata HR, Reynolds SH, doi:10.1016/j.devcel.2011.06.018
Fischer KA, Hatfield SD, Ruohola-Baker H 15. Kirilly D, Wang S, Xie T (2011) Self-maintained
(2006) Stem cells signal to the niche through escort cells form a germline stem cell differentia-
the Notch pathway in the Drosophila ovary. tion niche. Development 138(23):5087–5097.
Curr Biol 16(23):2352–2358. doi:10.1016/j. doi:10.1242/dev.067850
cub.2006.10.022 16. Margolis J, Spradling A (1995) Identification
13. Wang L, Li Z, Cai Y (2008) The JAK/STAT and behavior of epithelial stem cells in the
pathway positively regulates DPP signaling in Drosophila ovary. Development 121(11):
the Drosophila germline stem cell niche. 3797–3807
J Cell Biol 180(4):721–728. doi:10.1083/ 17. Gilboa L, Lehmann R (2006) Soma-germline
jcb.200711022 interactions coordinate homeostasis and growth
14. Losick VP, Morris LX, Fox DT, Spradling A in the Drosophila gonad. Nature 443(7107):
(2011) Drosophila stem cell niches: a decade 97–100. doi:10.1038/nature05068
Chapter 4
Morphometric Evaluation of the Spermatogonial

Stem Cell Distribution and Niche in Vertebrates
Paulo Henrique Almeida Campos-Junior, Guilherme Mattos Jardim
Costa, Gleide Fernandes de Avelar, Tânia Mara Segatelli, Samyra Maria
Santos Nassif Lacerda, Pedro Manuel Aponte, and Luiz Renato de França
Abstract
Morphometry is a classical quantitative method often used in biology to provide a data basis for functional
interpretations/interactions of a particular organ or system. Herein we took advantage of this valuable
approach to evaluate the spermatogonial stem cell niche using the horse testis and immunocytochemical
localization of GFRA1 [glial cell line-derived neurotrophic factor receptor produced by Sertoli cells)] as an
example. Using the NIH ImageJ free software, we describe in detail all the necessary steps to investigate this
specific and crucial microenvironment. Based on several recently published papers from our research group,
this approach has proved to be fast, simple, and adaptable to a wide range of species and has the potential
to be easily reproducible in different laboratories.
Key words Morphometry, Morphology, Testis, Spermatogenesis, Spermatogonial stem cell, Niche,
GFRA1, ImageJ
1 Introduction
A stem cell niche is considered a specialized microenvironment

that produces signals to control stem cell self-renewal, differentia-
tion, or survival in a correct balance specific to the needs of a given
stem cell system in a given tissue. In the testis, spermatogonial
stem cells (SSCs) are located in the seminiferous epithelium and in
addition to the physical support provided by Sertoli cells and the
basement membrane, the niche environment typically contains
intrinsic factors from key neighboring somatic cells (Sertoli,
Leydig, and peritubular myoid cells) and the basement membrane,
and also extrinsic factors originating from the vascular network
[1–6]. However, the specific roles of the different testis compo-
nents/cells constituting this important microenvironment have
not been clearly elucidated yet [6].
35
36 Paulo Henrique Almeida Campos-Junior et al.
Although the general testis cytoarchitecture is quite similar

among vertebrate species, there exist significant phylogenetic dif-
ferences in the distribution of the spermatogonial cells [7, 8].
Therefore, precise knowledge of this organ’s cytoarchitecture is
necessary in order to understand the relationship between SSCs
and other germ cells and somatic cells in the seminiferous tubule
[2–4]. Another important aspect to be considered is the morpho-
logical and/or phenotypical characterization of the SSCs and their
transit-amplifying (progenitors) cells [6, 9]. In this respect, the
quantity and distribution of nuclear heterochromatin is a feature
that usually allows the identification and characterization of the dif-
ferent spermatogonial cell types [10]. Furthermore, several early
spermatogonial markers are now available [2–6, 11]. Additionally,
long-term cell label-retaining approaches, such as BrdU or radio-
isotope incorporation, could be used for investigating particular
functional characteristics of rare, slowly cycling SSCs [2, 12, 13].
In this chapter we have taken advantage of the morphometric
approach to quantitatively evaluate SSC distribution and their niche.
This classical methodology has proved to be fast, simple, and adapt-
able to a wide range of species [2–4] and has the potential to be
easily reproducible in different laboratories. Hence, we will herein
describe all the necessary steps to evaluate the SSCs in the context
of their microenvironment in the testicular parenchyma, particularly
using a specific standard image processing software.
2 Materials
Because the success of the morphometric analysis employed here is

dependent on the correct identification of SSCs and their location,
the testis tissue sampling procedures and the use of adequately
preserved/fixed and embedded specimens are critical steps to be
properly considered [3, 4, 10] (please see Note 1). In this regard,
testis tissue fragments should be preferentially fixed in glutaralde-
hyde, paraformaldehyde, or Bouin’s solution and embedded in
araldite, glycol methacrylate, or paraplast. When morphological
criteria are solely used, glutaraldehyde/araldite is the best option.
However, the phenotypic SSC characterization usually requires
specific cell markers such as GFRA1, PLZF, Thy-1, Nanos-1,
Nanos-2, and CSF1r [1, 6, 11]. In this particular situation, fixa-
tion in Bouin’s solution and embedding in paraplast are often the
best choice. Here, we demonstrate the adequacy of GFRA1, a
receptor for the glial cell line-derived neurotrophic factor (GDNF)
produced by Sertoli cells [14], which is considered to be well con-
served among the different vertebrate classes (Fig. 1). Besides
that, as mentioned in the introduction, the identification of a par-
ticular category (subset) of SSCs can be performed using BrdU
incorporation (Fig. 1) (please see Notes 2 and 3).
Fig. 1 Seminiferous epithelium and spermatogonial morphology/phenotype in different vertebrate species
presenting cystic [zebrafish (Danio rerio) and bullfrog (Lithobates catesbeianus)] and non-cystic [turtle
(Kinosternon scorpioides); mouse (Mus musculus); collared peccary (Tayassu tajacu); and horse (Equus
caballus)] spermatogenesis arrangement. For each mentioned species, the first row (a–f) illustrates the semi-
niferous tubules at low magnification, whereas the second (g–l) and third (m–r) ones depict the spermatogo-
nial stem cells (SSCs; red arrowheads) characterized respectively according to morphological and phenotypic
criteria. As it can be observed, although the SSCs may present different shapes and sizes, these cells usually
have a low quantity of nuclear heterochromatin (g–l). Phenotypically, SSCs can be identified using GFRA1
expression as a functional marker (o, q, and r), whereas a subset of these cells (long-term label-retaining or
slow-cycling cells) could be characterized for instance by BrdU incorporation (m–n, and p) in zebrafish, bull-
frog, and mouse, respectively, at 7, 46, and 28 days post BrdU injection. Bars: a–f = 120 µm; g–r = 8 µm
All the necessary materials are listed below:

1. Histological slides previously prepared.
2. Photomicroscope equipped with a set of lenses to capture
good-quality pictures at different magnifications.
3. Image capture software (we use Cell^F, Olympus).
4. Image J software.
5. Statistical software.
3 Methods
3.1 Image 1. The images can be acquired using any system for image capture.
Acquisition The optimal resolution for further morphometrical analysis is
2,576 × 1,932 pixels.
2. Each image should be captured with a seminiferous tubule
cross section in a central position in order to observe the
neighboring components such as other tubules or interstitial
elements. In general, we use 200× magnification, but this
choice should be adapted according to the seminiferous tubule
diameter of the particular species investigated.
3.2 Spermatogonial 1. Download Image J free software, in the homepage: http://

Stem Cell Distribution rsbweb.nih.gov/ij/download.html.
Analysis 2. Open Image J.
3. As an illustration, the following steps will be described using

pictures of horse testis after GFRA1 immunostaining (please
see Note 3).
4. Open image that will be analyzed.
Morphometric Evaluation of the Spermatogonial Stem Cell Distribution… 39
5. Select the Paintbrush tool in the main menu.
6. Adjust the Brush width to 50 pixels, by clicking on the right

mouse button in the Paintbrush tool.
7. Mark the seminiferous tubule cross section central point and
the points in which the tubular circumference is in contact with
the different neighboring components that will be evaluated.
8. Select the Angle tool in the main menu.
9. Using the angle tool, join the points 1–0–2 to obtain the angle
of the specific evaluated region (e.g., tubule–tubule area).
10. Just after the angle acquisition, press “ctrl + M” to obtain the
respective value (e.g., 53.096°).
Morphometric Evaluation of the Spermatogonial Stem Cell Distribution… 41
11. Count the number of spermatogonial cells in this particularly

delimited area (e.g., 5 GFRA1-positive cells).
12. In the next step, the number of spermatogonial cells should be

divided by the angle area (expressed in degrees) in each region
(e.g., 5 cells/53.096° = 0.094).
13. The same analysis should be performed in all other considered
regions, up to the completion of 360° related to the total semi-
niferous tubule circumference.
14. Repeat all previously mentioned procedures in at least ten sem-
iniferous tubule cross sections, per each stage of the seminifer-
ous epithelium cycle (please see Note 4). Observe that in
mammals spermatogenesis is divided into different stages
according to the particular germ cell association [3, 4] (please
see Note 5).
3.3 Data Analysis 1. Calculate the mean cell density per area (degree) in each
region.
2. Apply the adequate statistical tests to compare different
regions.
4 Notes
1. All the necessary procedures for testis sampling and process-

ing, including fixation and embedding, that are crucial for
obtaining good-quality results, are described in details in sev-
eral publications from our research group [2–4, 15].
2. BrdU may be used at the concentration of 150 mg/kg of body
weight. Also, for properly dissolving BrdU it is important to
add DMSO first, and then the same volume of distilled water.
3. Please see recent publications for good immunohistochemistry

protocols for GFRA1 [3, 4] and BrdU [2, 13].
4. The minimum number of pictures that should be taken for
each germ cell association (we usually take 10) has to be calcu-
lated in order to decrease the coefficient of variation.
5. The accurate knowledge of spermatogenesis is crucial for the
better understanding of the spermatogonial stem cell biology
and niche. Therefore, previous knowledge of the testis mor-
phophysiology from the particular species under investigation
is necessary.
References
1. Hofmann MC (2008) Gdnf signaling pathways (2010) Spermatogenesis in fish. Gen Comp
within the mammalian spermatogonial stem cell Endocrinol 165(3):390–411
niche. Mol Cell Endocrinol 288:95–103 9. Yoshida S (2012) Elucidating the identity and
2. Nóbrega RH, Greebe CD, van de Kant H, behavior of spermatogenic stem cells in the
Bogerd J, França LR, Schulz RW (2010) mouse testis. Reproduction 144:293–302
Spermatogonial stem cell niche and spermato- 10. Chiarini-Garcia H, Meistrich ML (2008) High
gonial stem cell transplantation in zebrafish. resolution light microscopic characterization of
PLoS One 5:e12808 spermatogonia. Methods Mol Biol 450:95–107
3. Campos-Junior PHA, Costa GMJ, Lacerda 11. Phillips BT, Gassei K, Orwig KE (2010)
SMSN, Rezende-Neto JV, de Paula AM, Spermatogonial stem cell regulation and sper-
Hofmann MC, França LR (2012) The matogenesis. Philos Trans R Soc Lond B Biol
spermatogonial stem cell niche in the Sci 365:1663–1678
collared peccary (Tayassu tajacu). Biol Reprod 12. Huckins C (1971) The spermatogonial stem
86(155):1–10 cell population in adult rats. 3. Evidence for a
4. Costa GMJ, Avelar GF, Rezende-Neto JV, long-cycling population. Cell Tissue Kinet
Campos-Junior PHA, Lacerda SMSN, Andrade 4:335–349
BS, Thomé RG, Hofmann MC, Franca LR 13. Ehmcke J, Schlatt S (2008) Identification and
(2012) Spermatogonial stem cell markers and characterization of spermatogonial subtypes
niche in equids. PLoS One 7:e44091 and their expansion in whole mounts and tis-
5. Lacerda SMSN, Aponte PM, Campos-Junior sue sections from primate testis. Methods Mol
PHA, Costa GMJ, Segatelli TM, Silva MA, Biol 450:109–118
França LR (2012) An overview on spermato- 14. Meng X, Lindahl M, Hyvönen ME, Parvinen
gonial stem cell physiology, niche and trans- M, de Rooij DG, Hess MW, Raatikainen-
plantation. Anim Reprod 9:798–808 Ahokas A, Sainio K, Rauvala H, Lakso M,
6. Oatley JM, Brinster RL (2012) The germline Pichel JG, Westphal H, Saarma M, Sariola H
stem cell niche unit in mammalian testes. (2000) Regulation of cell fate decision of
Physiol Rev 92:577–595 undifferentiated spermatogonia by GDNF.
7. Fawcett DW, Neaves WB, Flores MN (1973) Science 287:1489–1493
Comparative observations on intertubular 15. Leal MC, Cardoso ER, Nóbrega RH, Batlouni
lymphatics and the organization of the intersti- SR, Bogerd J, França LR, Schulz RW (2009)
tial tissue of the mammalian testis. Biol Reprod Histological and stereological evaluation of
9:500–532 zebrafish (Danio rerio) spermatogenesis with
8. Schulz RW, França LR, Lareyre JJ, Le Gac F, an emphasis on spermatogonial generations.
Chiarini-Garcia H, Nóbrega RH, Miura T Biol Reprod 81:177–187
Chapter 5
In Vitro Construction of 2D and 3D Simulations

of the Murine Hematopoietic Niche
Brahmananda Reddy Chitteti, Monique Bethel, Sherry L. Voytik-Harbin,
Melissa A. Kacena, and Edward F. Srour
Abstract
Hematopoietic stem cells (HSC) undergo multilineage differentiation or self-renewal to maintain normal
hematopoiesis and to sustain the size of the HSC pool throughout life. These processes are determined by a
complex interplay of molecular signals between HSC and other cellular components such as osteoblasts
(OB), stromal cells, endothelial cells, and a number of extracellular matrix (ECM) proteins. Through changes
in its physical properties within the bone marrow (BM) microenvironment, collagen, which is one of the
most critical ECM proteins, can modulate HSC function and maintenance of the competence of the hema-
topoietic niche (HN). At present, there is no consensus as to how different cellular elements of the niche
collaborate and interact to promote HSC self-renewal or differentiation to maintain hematopoiesis.
Deciphering these interactions and the impact of mechanical properties of the collagen microstructures
within the HN has critical clinical implications in the areas of stem cell homing, engraftment, and mainte-
nance of HSC function. In this chapter, we describe several of the in vitro methodologies for establishing and
maintaining HSC in vitro including the isolation of OB, stromal cells, and hematopoietic progenitor cells, as
well as the establishment of both two-dimensional (2D) and three-dimensional (3D) coculture systems.
Key words Hematopoietic stem cells, Osteoblasts, Stromal cells, Stem cell niche, Collagen matrix
1 Introduction
Lifelong production and maintenance of all blood cells are sustained

by a group of highly specialized cells known as hematopoietic stem
cells (HSC) [1–3]. In adults, HSC are believed to reside in deep
dormancy within the bone marrow (BM) microenvironment in
specialized areas of the marrow called the “hematopoietic niche
(HN)” [4, 5]. Within the HN, stem cells perform two unique
functions: multilineage differentiation and self-renewal [5, 6].
These poorly understood functions enable HSC to maintain
steady-state hematopoiesis and to restore normal hematopoietic
functions following BM transplantation [7].
43
44 Brahmananda Reddy Chitteti et al.
The concept of the HN was first introduced by Schofield [8].

Since then, a more detailed picture of the HN emerged in which two
niches, the endosteal and the vascular niche, are now distinguished
[9]. It is generally accepted that quiescent HSC reside in the endos-
teal region in close proximity to osteoblasts (OB) where they receive
signals that maintain their quiescence [10–12], while more active
HSC reside in the vascular niche and are primed to respond quickly
to hematopoietic stress and entry into the circulation [13, 14]. In
addition to interacting with OB and endothelial cells, HSC also
interact with a vast assembly of other cell types within the BM known
collectively as stromal cells. Stromal cells is a collective term encom-
passing many cell types including epithelial cells, endothelial cells,
reticular cells, macrophages, and adipocytes [15]. Stromal cells and
a large number of soluble cytokines and growth factors in the HN
collaborate to control recruitment of stem cells into active phases of
cell cycle and subsequent decisions to self-renew or differentiate.
However, both cellular and molecular mechanisms by which interac-
tions between HSC and elements of the niche control and direct
HSC functions remain unresolved [16].
Besides the impact of cells of the HN on HSC function, the
extracellular matrix (ECM) confers physical attachment, organiza-
tion, and structural support to the HSC microenvironment. The
complex collection of cell types and ECM represents a 3D, visco-
elastic connective tissue or matrix within the cavities of bones.
Studies have documented the beneficial effects of culturing HSC
on tissue culture plastic coated with laminin or fibronectin [17] or
undefined BM ECM extracts [18]. However, the physiologic rel-
evance of such ECM signaling within the context of 3D structural-
mechanical complexity and how it modulates the HSC fate are yet
to be elucidated. In order to understand the impact of various cel-
lular elements and biophysical cues inherent to the ECM on HSC,
we describe in this chapter methods for (1) isolation and character-
ization of osteoblasts, stromal cells, and HSC; (2) preparation of a
2D coculture system; and (3) creation of 3D coculture system
within a polymerizable, collagen-fibril matrix.
2 Materials
2.1 Osteoblast 1. Collagenase solution: 25 mL of 1× phosphate-buffered saline

Preparation (PBS) with 200 U/mL of Type II collagenase (final concentra-
tion, Worthington) and 200 μL/mL N(a)-tosyl-lys chloromethyl
ketone, hydrochloride (final concentration). Filter it using
0.2 μM filter and pre-warm the solution in 37 °C water bath.
2. 4 mM Ethylene diamine tetra acetic acid (EDTA), pre-warm
the solution in a 37 °C water bath.
3. 70 % Ethanol.
In Vitro Construction of 2D and 3D Simulations of the Murine Hematopoietic Niche 45
4. Sterilized T pins.
5. Sterilized filters (Mesh Screens—297 μm or Sweeny filter).
6. Complete alpha minimum essential medium (αMEM) consist-
ing of αMEM supplemented with 10 % fetal calf serum (FCS),
1 % penicillin/streptomycin (P/S), and 1 % L-glutamine.
7. 1× PBS.
2.2 Stromal Cell 1. 25 or 75 cm2 tissue culture flasks or tissue culture dishes.
Preparation 2. Ficoll purchased as a ready-to-go solution. Make sure that the
bottle of Ficoll is warmed up to room temperature before using.
3. Complete Iscove’s modified Dulbecco’s medium (IMDM)
consisting of IMDM supplemented with 10 % FCS, 1 % P/S,
and 1 % L-glutamine.
4. Heparin medium consisting of Hank’s Balanced Salt Solution
(HBSS) medium supplemented with 1 % P/S and 20 U/mL
heparin.
5. β-Mercaptoethanol prepared at 2 × 10−2 M in complete IMDM
(100×).
6. Methylprednisolone or hydrocortisol prepared at 2 × 10−3 M in
complete IMDM (100×).
2.3 Hematopoietic 1. Ficoll purchased as a ready-to-go solution. Make sure that the
Stem Cells bottle of Ficoll is warmed up to room temperature before using.
2. Complete IMDM consisting of IMDM supplemented with
10 % FCS, 1 % P/S, and 1 % L-glutamine.
3. Heparin medium consisting of HBSS medium supplemented
with 1 % P/S and 20 U/mL heparin.
4. PBS solution supplemented with 1 % FCS.
5. Fluorochrome-labeled primary antibodies for immunostain-
ing. These antibodies may vary in number and specificities
depending on the phenotypic definition of the cells that will be
identified and isolated by flow cytometric cell sorting.
2.4 Preparation of 1. 12- or 24-well tissue culture plates.

2D Coculture System 2. Complete medium consisting of IMDM supplemented with
3. 1:1 mix of IMDM and αMEM supplemented with 10 % FCS,
1 % P/S, and 1 % L-glutamine.
2.5 Preparation of 1. Positive displacement pipette.

3D Coculture System 2. 5 and 15 mL sterile centrifuge tubes.
3. 24-well plates.
4. Type I collagen oligomers acid-extracted and purified from the

dermis of porcine skin [19, 20]. Collagen concentration is
determined using a Sirius Red (Direct Red 80) assay as previ-
ously described [21]. Collagen formulations are standardized
based upon purity as well as polymerization potential [20].
Here, polymerization potential is defined as the relationship
between shear storage modulus (G’; stiffness) of polymerized
matrices and collagen content of the polymerization reaction.
5. Collagen polymerization reagents: 0.01 N hydrochloric acid
(HCl), 10× PBS (1× PBS has 0.17 M total ionic strength and
pH 7.4), 0.1 N sodium hydroxide (NaOH), and 13.57 mM
calcium chloride (CaCl2) in 0.01 N HCl.
6. Complete medium consisting of IMDM supplemented with
2.6 Harvesting 1. Extraction medium: Collagenase, Type IV (Worthington) at

Cells from 3D concentration of 500 U/mL and dispase (Worthington) at
Coculture System concentration of 1–2.4 U/mL in IMDM medium.
2. FCS.
3. 1× Gibco TripLE trypsin.
3 Methods
3.1 Osteoblast 1. In a sterile environment (e.g., biosafety cabinet) euthanize

Preparation 2–3-day-old mouse pups (see Note 1).
2. Make a sterile stage by wrapping a foam board with a sterilized
paper towel.
3. Prepare 3 small (50 mL) beakers of 70 % ethanol for sequential
sterilization of pups.
4. Euthanize pups and then sterilize the bodies by soaking pups
(head down) sequentially through the 3 beakers of 70 %
ethanol. Decapitate the pups with sterile scissors and place the
heads on the sterile stage.
5. Fix all heads on the sterile stage with sterilized T pins. The pin
should be placed through the nose with the cranium up with the
back of the head toward the opening in the biosafety cabinet.
6. Lift the skin at the back of the skull, separate it from the calvaria
with a small scissors, cut at both sides close to ears, and fold it
forward.
7. Make cuts on the calvarial bone at base part close to neck, both
sides, and between the eyes.
8. Lift the calvaria, remove any soft tissue, and place calvaria in a
10 mL complete αMEM medium in a sterile tissue culture dish.
9. Gently clean the calvaria by lightly scraping off soft tissue

membranes using the back of curved forceps.
10. Collect all the calvariae similarly from all of the pups.
11. Cut all of the calvariae in half and place them into a 50 mL
conical tube.
12. Add 10 mL 4 mM EDTA into the tube and incubate for
10 min, shaking at 37 °C (all shaking is vigorous so that the
calvariae/liquid are moving in the tube, 90+ rpm pending
shaker model).
13. Remove EDTA using a cotton-plugged pipette, wash once
with 5 mL PBS, and remove PBS also by using a cotton-
plugged pipette.
14. Repeat steps 12 and 13 once.
15. Add 5 mL of collagenase solution into the tube and incubate
for 10 min, shaking at 37 °C. Shake by hand once during the
incubation period.
16. Remove collagenase using a cotton-plugged pipette, wash once
with 5 mL PBS, and remove PBS using a cotton-plugged pipette.
17. Repeat steps 15 and 16 once.
18. Add 5 mL collagenase into the tube and incubate for 15 min,
shaking at 37 °C. Shake by hand once during the incubation
period. Collect collagenase solution using a cotton-plugged
pipette, and filter the solution into a 50 mL tube (Sweeny or
mesh filter; please see Note 2). Wash calvariae once with 5 mL
PBS, collect the PBS, and filter it into the same collagenase
collection tube.
19. Repeat step 18 twice.
20. Centrifuge collagenase collection tube at 500 × g for 6 min.
21. Remove the supernatant, and resuspend the cells in 10 mL
complete αMEM.
22. Let the tube set for 1–2 min and transfer the supernatant to
another tube (to remove any debris).
23. Repeat the above step once if needed.
24. Count cells by differential trypan blue staining.
25. It is normally expected to obtain 0.5–2e6 OB per pup.
26. This protocol results in approximately a 95 % pure population
of OB cells or OB precursor cells [22].
27. If of interest, flow cytometric sorting can then be utilized to
isolate phenotypically defined populations of OB lineage cells
[23]. A typical population used is Lineage (CD45, CD31,
Ter119)− Sca-1− CD51+ Osteopontin+ CD166+. Figure 1 is
a representative dot plot from calvarial OB stained with these
monoclonal antibodies that can be used for the identification
Fig. 1 A representative dot plot showing the 2-day calvarial osteoblast surface phenotype by flow cytometry.
83 % of osteoblasts prepared as described in Subheading 3.1 are lineage (CD45, CD31, and Ter119) negative
and Sca-1 negative. Among these lineage- and Sca1-negative cells, 90.1 % cells are CD51 positive, 95 % cells
are osteopontin positive, and 34 % cells are CD166 (ALCAM) positive
Fig. 2 (a) A representative figure showing osteoblasts that were isolated from the 2-day calvariae of C57Bl/6
pups as described in Subheading 3.1 and were cultured for 4 days. (b) A representative figure of stromal cells
prepared from adult C57Bl/6 mice and cultured for 4 weeks as described in Subheading 3.2. Please note that
stromal cells shown in this figure are not yet at a monolayer stage
of classes of OB. Please follow general staining protocols

presented below in Subheading 3.3 for the isolation of HSC.
Figure 2a is a representative micrograph of OB in culture.
28. For the isolation of OB from long bones please see Note 3.
3.2 Stromal Cell 1. Euthanize a 6–10-week-old mouse and sterilize with 70 %

Preparation ethanol.
2. Collect all four limbs into heparin medium.
3. Strip bones of muscle and soft tissue using sterile gauze.
4. Flush the bone very thoroughly using 27G needle with
8–10 mL of heparin medium. To accomplish this, cut long
bones in half keeping the epiphyses intact at the end of each

half of the long bones. With sterile forceps, grab a piece of the
cut bone and introduce the needle into the shaft of the bone
aiming toward the epiphysis. Inject 1–2 mL of heparin medium
into the bone while retracting and pushing the needle into the
bone shaft. Make sure that the tip of the needle reaches the
epiphysis to ensure that the epiphysis is thoroughly washed
with the injected medium. If necessary, inject more medium to
release the majority of the BM cells and eject them out of the
bone shaft. A well-flushed bone will normally lose the red color
of the BM and become almost translucent.
5. Alternatively bones can be crushed in the heparin medium
using a mortar and pestle and then cells can be transferred into
a 50 mL conical tube by passing through a 40 μm filter.
6. Using Ficoll, separate the low-density BM cells from RBC and
other cells contained in the BM. This is accomplished by layer-
ing flushed BM cells on top of Ficoll and centrifuging the mix
for 30 min at room temperature. Collect low-density cells at
the interface of medium and Ficoll. Use repeated slow aspira-
tion of the cells at interface to ensure the collection of all of the
cells (for details on how to Ficoll BM cells please see Note 4).
7. Place the cells into a 50 mL tube, and fill the tube with HBSS
medium to dilute the Ficoll as much as possible.
8. Centrifuge at 500 × g for 10 min at 4 °C. Decant supernatant.
9. Resuspend the cells in complete IMDM medium at a concen-
tration of 1.0e6 cells per mL.
10. The general rule for establishing stromal cultures is to use 1.0e6
cells per 2 cm2 of any flask used. Calculate the volume of cells
(at 1.0e6 cells/mL) required for the number of flasks/wells/
dishes to be prepared (total area to be seeded in cm2 divided by
2 = volume of cell suspension required) (see Note 5).
11. For each 1 mL of complete IMDM medium with cells in step 10,
add 10 μL of 100× β-mercaptoethanol solution and 10 μL of
either 100× methylprednisolone or hydrocortisol solution.
12. Incubate cultures at 37 °C, 95 % humidity, and 5 % CO2.
13. During the next 24–48 h, very gently swirl the flask 2–3 times
while it is flat on the working surface of the hood and tilt the
container to “drain” the medium to one side. Very gently aspi-
rate the bulk of the medium to remove non-adherent cells.
14. Replace the same volume of medium removed in step 13 and
make sure that the new medium contains β-mercaptoethanol
and methylprednisolone/hydrocortisol solution as in step 11.
15. Repeat steps 13 and 14 weekly thereafter. A confluent mono-
layer of stromal cells should develop 4–6 weeks later.
16. A representative Figure of a 4-week stromal culture, not yet as
a full monolayer, is shown in Fig. 2b.
3.3 Hematopoietic 1. Repeat steps 1 through 9 described above in Subheading 3.2

Stem Cell Preparation describing stromal cell preparation.
2. Using tubes compatible with the available flow cytometric cell
sorting equipment, prepare tubes with 2–5 × 10e5 cells each
for isotype, single-color positive controls, and fluorescence
minus-one (FMO) controls. Fill these tubes with 2–3 mL PBS
supplemented with 1 % FCS.
3. Prepare 1 tube with a sufficient number of low-density BM cells
to yield the required number of isolated enriched HSC popula-
tions. The number of cells to be used depends on the degree of
characterization of these cells and the final size of the group of
cells to be selected.
4. As an example here, we will cover what is required to isolate
Lineage-Sca1+ ckit+ (LSK) cells. These cells are identified by
three sets of markers. Lineage markers (a cocktail of lineage-
specific markers that identifies differentiated hematopoietic
cells within specified cell lineages such as T cells (CD3, CD4,
and CD8), B cells (B220), and myeloid cells (GR-1, Mac1,
CD11b, CD14)), Sca-1, and C-kit or CD117. The lineage
marker cocktail can vary in composition, but it should at least
cover the three lineages listed above.
5. For this combination, a total of seven control tubes are required
and one “sorting” sample stained with all the antibodies
simultaneously.
6. Add to tube 1 all the isotype control antibodies. Add to tube 2
all the lineage antibodies (this cocktail can be designed to cover
any combination required and all antibodies used should be
conjugated to the same fluorochrome to allow for the selection
of negative cells based on the collective positive signal from all
markers combined). Add to tube 3 the Sca-1 antibody. Add to
tube 4 the CD117 antibody. Add to tube 5 all the lineage
markers plus Sca-1 and the isotype antibody that is matched to
the fluorochrome to which CD117 is conjugated. This is the
FMO for CD117. Stain tubes 6 and 7 in similar fashion to tube
5 to generate the FMO controls for Sca-1 and the lineage
markers. Tube 8 should be stained with all the lineage cocktail
antibodies, Sca-1 and CD117.
7. Stain cells on ice for 20 min.
8. Wash cells once with PBS supplemented with 1 % FCS.
9. Isolate LSK cells by flow cytometric cell sorting.
3.4 Preparation of 1. Plate either freshly prepared or cultured OB or cultured stro-

2D Coculture System mal cells either individually or in combinations at a frequency
of 40,000 OB cells or 100,000 stromal cells, respectively, per
well in a 12-well plate.
2. The next day, add HSC (1,000 LSK cells) to the OB and/or
stromal cell cultures.
3. All cultures are then supplemented with a cocktail of cytokines
containing recombinant murine SCF and IL3 (10 ng/mL),
IGF1 and TPO (20 ng/mL), IL6 and Flt3 (25 ng/mL), and
OPN (50 ng/mL). Maintain cultures for 1 week in medium
consisting of 1:1 mix of IMDM and αMEM supplemented with
10 % FCS, 1 % Pen/Strep, and 1 % L-glutamine. On day 5
replenish the cultures again with the same cytokine mixture.
4. It should be noted that by day 7 cultures containing LSK cells
and cytokines have numerous hematopoietic cells making visu-
alization of OB and/or stromal cells difficult. Importantly, OB
and/or stromal cells should remain healthy throughout the
coculture period (e.g., they are not peeling off).
5. Hematopoietic cells are typically harvested on day 7 and
counted. Fold increase in the number of cells derived from
LSK cells is calculated relative to d0 count of 1,000. At this
time cells can be analyzed by standard in vitro assays such as
phenotyping and colony forming assays, or they can be used in
in vivo transplantation assays [24, 25].
3.5 Preparation of 1. All steps for 3D coculture system preparation should be per-
3D Coculture System formed on ice (4 °C) unless otherwise mentioned.
2. To accurately pipette the viscous collagen solution, a positive
displacement pipette should be used; please see Note 6.
3. Transfer collagen solution into a 15 mL tube and keep it on
ice. The volume of collagen solution is chosen based on the
desired final collagen concentration of the polymerization
reaction or stiffness (G’) of the polymerized matrix. Refer to
Table 1 for polymerization reaction reagents and recipes (reci-
pes are based on collagen polymerization potential).
4. Add sterile 0.01 N HCl to the collagen solution. Invert several
times to mix well.
5. Neutralize collagen solution to achieve neutral pH (7.4) by add-
ing sequentially 10× PBS, sodium hydroxide, and CaCl2. Invert
to mix after the addition of each component. Maintain the
neutralized collagen solution on ice until ready to polymerize.
6. Aliquot cell solutions containing specified number of stromal
cell, OB, and HSC into a 50 ml tube.
7. Centrifuge cell solution at 500 × g for 10 min at 4 °C. Decant
the supernatant. Vortex gently to disrupt the cell pellet.
8. Transfer neutralized collagen solution to cell pellet and mix by
inverting. It is important to achieve a homogenous distribu-
tion of cells within the collagen solution.
Table 1
The amount of collagen and other reagents required to make a particular stiffness (Pa)
three-dimensional scaffolds
Reagent amounts (µL)

Final collagen
concentration Final matrix Final Collagen
(mg/mL) stiffness (Pa) volume HCl (6.3 mg/mL) PBS NaOH CaCl2
0.38 10 1,000 714 61 100 100 25
1.31 100 1,000 567 208 100 100 25
1.81 200 1,000 488 287 100 100 25
2.78 500 1,000 333 442 100 100 25
3.48 800 1,000 223 552 100 100 25
3.87 1,000 1,000 161 614 100 100 25
4.22 1,200 1,000 104 671 100 100 25
9. Aliquot the cell suspension into wells of a tissue culture well

plate (for example, 500 μL/well for a 24-well plate).
10. Place the well plate at 37 °C for approximately 15 min to allow
polymerization.
11. Immediately following polymerization, add 1–1.5 mL com-
plete medium to each well.
Incubate plate at 37 °C within a humidified atmosphere of 5 %
CO2 in air for the desired study duration (typically 5–7 days).
A photomicrograph of a 200 Pa 3D collagen matrix is shown
in Fig. 3.
12. Figure 4 shows HSC cultured within a 200 Pa collagen matrix
for 7 days.
3.6 Harvesting 1. Prepare the cell extraction solution (for example, 1 mL per
Cells from 3D 500 μL tissue construct). Filter cell extraction solution through
Coculture System a sterile 0.2 μM syringe filter. Warm solution to 37 °C imme-
diately prior to use.
2. Add 1 mL of cell extraction solution to a 15 mL sterile conical
tube.
3. Using a sterile forceps, transfer tissue construct from well plate
to tube containing cell extraction solution.
4. Shake tubes at 120 rpm, 37 °C, for 20 min. Invert tubes sev-
eral times every 5–10 min.
5. Add an equal volume of complete medium (Subheading 2.3
above) and pipette up and down gently to ensure complete
digestion of the collagen construct.
Fig. 3 Fibril microstructure of collagen oligomer matrix polymerized at collagen

concentration of 1.5 mg/mL as visualized using confocal microscopy
Fig. 4 A representative figure of murine HSC (lineage− Sca1+ cKit+ or LSK cells)
proliferating in 200PA collagen constructs that are prepared as described in
Subheading 3.4
6. Centrifuge the mixture at 500 × g for 5 min at room

temperature.
7. Decant the supernatant and vortex the pellet gently.
8. Add 5 mL of complete medium (Subheading 2.3 above) and
pipette up and down gently. Repeat centrifugation and vortex
step as above.
9. Add 100 μL Gibco TripLE to the cells and pipette up and
down gently.
10. Shake at 120 rpm, 37 °C, for 15 min. Invert tubes several
times every 5 min.
11. Add 400 μL complete medium to stop the trypsin and enu-
merate the cells with differential trypan blue staining.
4 Notes
1. 1–5-day-old pups may be used, but best preparations are made

from 2- to 3-day-old pups.
2. During the OB preparation, the collected collagenase diges-
tions/PBS washes should be filtered through a 297 μM mesh
or Sweeny filter (not 0.2 μm filter) to eliminate bone pieces.
3. OB from long bones can be obtained following the similar
procedure as mentioned in Subheading 3.1 after flushing the
BM out. Long bones need to be cut with small scissors into
pieces less than 1 mm before collagenase digestions. The bone
segments subjected to 2 consecutive collagenase digestions
(30 min and 1 h) and cells are collected after each cycle, and
pooled.
4. Ficolling: Suspend BM cells in 30 mL medium or PBS/tube in
a 50 mL conical tube. Carefully underlay 13.5 mL Ficoll
beneath the cell suspension by inserting a 10 mL pipette at the
bottom of the tube and releasing the Ficoll slowly allowing it
to settle below the cell suspension. Alternatively, the cell sus-
pension can be overlaid on top of the Ficoll. As mentioned
above, the volume of Ficoll required for a 50 mL conical tube
is 13.5 mL and if the cells are layered in a 15 mL conical tube,
then only 4 mL of Ficoll are used (delivered in a 5 mL pipette
to avoid spilling). In general it is easier to underlay Ficoll in a
50 mL tube and to overlay cells (on top of 4 mL Ficoll) in a
15 mL tube.
5. Although seeding fewer cells may result in a monolayer,
1e6cells/2 cm2 is the optimal seeding density. Stromal cell lay-
ers can be prepared from both total low-density BM cells as
well as low-density BM cells depleted of hematopoietic stem
and progenitor cells.
6. To accurately pipette the viscous collagen solution a positive

displacement pipette should be used. In our studies, we used
Gilson Microman M1000 pipette with appropriate tips
(Microman, Gilson, Inc., Middleton, WI).
Acknowledgments
The authors thank the operators of the Indiana University Melvin

and Bren Simon Cancer Center Flow Cytometry Resource Facility
for their outstanding technical help and support. This work was
supported in part by grant NHLBI HL55716 (E.F.S.). Indiana
University is an NIDDK designated Center of Excellence in
Molecular Hematology (NIDDK P01 DK090948). M.B. is sup-
ported by an NHLBI training grant (T32 HL007910-13). The
Flow Cytometry Research Facility is partially funded by NCI P30
CA082709.
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Chapter 6
Isolation of Embryonic Hematopoietic Niche Cells

by Flow Cytometry and Laser Capture Microdissection
Daisuke Sugiyama and Tatsuya Sasaki
Abstract
Hematopoietic stem cells (HSCs) can differentiate into several types of hematopoietic cells, such as
erythrocytes, megakaryocytes, lymphocytes, neutrophils, or macrophages, and also undergo self-renewal
to sustain hematopoiesis throughout an organism’s lifetime. HSCs emerge and expand during mouse
embryogenesis. HSC regulation is governed by two types of activity: intrinsic activity programmed pri-
marily by cell autonomous gene expression, and extrinsic factors, which originate from the so-called
niche cells surrounding HSCs. Previously, we reported that endothelial niche cells regulate HSC genera-
tion at aorta-gonad-mesonephros region and placenta, and that hepatoblastic niche cells regulate HSC
differentiation in mouse embryonic liver. In the course of those studies, we employed immunohisto-
chemistry, flow cytometry, and the laser capture microdissection system to assess embryonic regulation
of the mouse hematopoietic niche.
Key words Hematopoietic stem cells, Niche cells, Embryo, Visualization, Laser capture microdissection
1 Introduction
During mouse embryogenesis, hematopoiesis begins in the yolk

sac (YS), producing mainly primitive erythroid cells at 7.5 days
post coitum (dpc) [1–5]. Shortly thereafter, definitive myelo-
erythroid progenitor cells appear in the YS (around 8.25 dpc) and
then seed the fetal liver [2, 6]. Although there is controversy over
where hematopoietic stem cells (HSCs) are generated—in the
extraembryonic mesoderm at the YS or in the intraembryonic
para-aortic splanchnopleural mesoderm (P-Sp)/aorta-gonad-
mesonephros (AGM) region—recent studies suggest that both
regions contain HSCs capable of reconstituting adult bone marrow
hematopoiesis, but the YS likely contributes to HSC generation to
a lesser extent [7–12]. In addition, HSCs are generated in the
placenta, likely independent of the YS and P-Sp/AGM region
[13–15]. These HSCs are then thought to circulate and colonize
fetal liver, where HSC expansion and differentiation occur, through
57
58 Daisuke Sugiyama and Tatsuya Sasaki
interaction between integrin-beta-1 and extracellular matrix factors

[16–22]. HSCs then temporarily reside in fetal spleen and finally
shift to fetal bone marrow [1–5]. Thus, the YS, P-Sp/AGM region,
placenta, liver, spleen, and bone marrow are regarded as hemato-
poietic organs. There, hematopoiesis is regulated extrinsically by
“niche cells” located adjacent to hematopoietic cells. Understanding
mechanisms governing niche cell regulation of HSCs and other
hematopoietic cells will enable us to improve HSC transplanta-
tion and hematopoietic cell transfusion for future clinical thera-
pies. For this purpose, we developed a method to assess cellular
interactions by observing cell morphology and using immunohis-
tochemistry to evaluate marker expression [15, 21–23]. We
improved confocal imaging by using 20 μm cryosections rather
than thinner conventional ones (7–10 μm). Using this method,
hematopoietic cell clusters containing HSCs, as defined by HSC
markers such as CD31, CD34, and c-Kit, were successfully visual-
ized in the P-Sp/AGM region and placenta by confocal micros-
copy, enabling identification of niche cells that may regulate HSC
generation [15, 23]. Furthermore, c-Kit-positive HSCs and
hematopoietic progenitors in liver were also visualized to identify
niche cells that may regulate their proliferation and differentia-
tion, particularly along the erythroid lineage [21, 22]. We then
employed flow cytometry to isolate endothelial niche cells expressing
CD31 and CD34 from both the P-Sp/AGM region and placenta
and hepatoblastic niche cells expressing DLK-1 from liver for
expression analysis and culture [15, 21, 22]. To further isolate spe-
cific cell compartments in tissue sections under the microscope, we
used the laser capture microdissection (LCM) system to collect
niche cells surrounding hematopoietic cell clusters that expressed
c-Kit. This approach was successful, despite the fact that investiga-
tion of the embryonic niche has been impeded by a lack of
markers.
2 Materials
2.1 Immunohisto- 1. Mouse embryos.

chemistry 2. Primary antibody solution for the AGM region and placenta:
PBS containing 1 % BSA with appropriate dilutions of the fol-
lowing primary antibodies: goat anti-mouse Kit (1:500; R&D
Systems), rat anti-mouse CD31 (1:500; BD Biosciences), rat
anti-mouse CD34 (1:500; BD Biosciences). For fetal liver,
anti-mouse DLK-1 Ab (1:250; MBL), and anti-mouse c-Kit
Ab (1:500; R&D Systems) (see Note 1).
3. Secondary antibody solution for the AGM region and pla-
centa: PBS containing 1 % BSA with appropriate dilutions of
the following secondary antibodies: Alexa Fluor 488 donkey
Isolation of Embryonic Hematopoietic Niche Cells 59
anti-rat IgG (1:300; Invitrogen) and Alexa Fluor 568 donkey

anti-goat IgG (1:300; Invitrogen) plus TOTO-3 (1:1,500;
Invitrogen) to stain nuclei. For fetal liver, donkey anti-goat
IgG-Alexa568, donkey anti-rat IgG-Alexa488 (all from Life
Technologies), and TOTO-3 (1:1,500; Invitrogen).
4. Fixative: 2 % paraformaldehyde in PBS.
5. 30 % sucrose in PBS.
6. Liquid nitrogen.
7. OCT compound (SAKURA, Tokyo, Japan).
8. Glass slides (Matsunami, Osaka, Japan).
9. Coverslips.
10. Fluorescence mounting medium (Dako Corporation).
11. FluoView 1000 confocal microscope (Olympus).
2.2 Flow Cytometry 1. Mouse embryos.

2. 21-gauge needle (Terumo).
3. Collagenase solution: 1 mg/ml collagenase in medium supple-
mented with 10 % fetal bovine serum (see Note 2).
4. 40 μm cell strainer (BD Pharmingen).
5. Lysing buffer: BD Pharm Lyse Lysing Buffer (BD Pharmingen).
6. Propidium iodide (PI) buffer (Invitrogen).
7. Fluorescence-conjugated antibodies: The following antibodies
are used to isolate endothelial and mesenchymal cell popula-
tions: FITC-conjugated anti-mouse Ter-119 (eBioscience),
PE-conjugated anti-mouse CD31 (BD Biosciences), APC-
conjugated anti-mouse c-Kit (BD Biosciences), PE-Cy7-
conjugated anti-mouse CD45 (BioLegend), and Pacific
Blue-conjugated anti-mouse CD34 (eBioscience). The follow-
ing antibodies are used to isolate hepatoblasts and sinusoid
endothelial cell populations: FITC-conjugated anti-mouse
DLK-1 Ab (MBL), PE-conjugated anti-mouse LYVE-1 Ab
(MBL), APC-conjugated anti-mouse CD31 Ab (Biolegend),
PE-Cy7-conjugated anti-mouse CD45 Ab (eBioscience), and
PE-Cy7-conjugated anti-mouse Ter119 Ab (eBioscience).
8. Flow cytometry: FACS Aria cell sorter (BDIS).
9. RNA later (Ambion).
2.3 Laser Capture All solutions are made RNAse-free by treatment with diethyl
Microdissection pyrocarbonate.
1. Mouse embryos.
2. Primary antibody solution: PBS containing 1 % BSA with goat
anti-mouse Kit (1:500; R&D Systems).
3. Secondary antibody solution: PBS containing 1 % BSA with

Alexa Fluor 568 donkey anti-goat IgG (1:300; Invitrogen).
4. Liquid nitrogen.
5. OCT compound (SAKURA, Tokyo, Japan).
6. Glass slides (Matsunami, Osaka, Japan).
7. Diethyl pyrocarbonate (Wako, Osaka, Japan).
8. Cryostat: Leica CM1900 UV cryostat.
9. LCM system: ArcturusXT™ Laser Capture Microdissection
System (Molecular Devices).
10. RNA extraction buffer: RNA extraction buffer included in the
Pico Pure™ RNA Isolation Kit (Molecular Devices).
3 Methods
3.1 Immunohisto- Carry out all procedures at room temperature unless otherwise
chemistry specified.
1. Dissect out mouse embryos and fix in 2 % paraformaldehyde in
PBS overnight.
2. Equilibrate embryos in 30 % sucrose in PBS overnight.
3. Wash them in PBS.
4. Embed embryos in OCT compound and freeze in liquid
nitrogen.
5. Slice tissues at 20 μm thickness using a cryostat and transfer to
glass slides.
6. Dry sections thoroughly (see Note 3).
7. Wash in PBS 3 times for 10 min each.
8. Block in 1 % BSA in PBS for 1 h.
9. Incubate sections with primary antibody solution at 4 °C
overnight.
10. Wash in PBS 3 times for 30 min each.
11. Incubate with secondary antibody solution at room tempera-
ture for 30 min (see Note 4). After adding secondary antibody
solution to glass slides, carry out all procedures in a darkroom
to preserve fluorescence.
12. Wash samples in PBS 3 times for 30 min each.
13. Mount samples on coverslips using fluorescence mounting
medium. Wait until medium dries thoroughly before analyzing
samples (see Note 5).
14. Evaluate using a confocal microscope (Fig. 1).
Fig. 1 Confocal images of hematopoietic organs during embryogenesis. Tissues sliced at 20 µm were stained
with antibodies and observed under confocal microscopy. (a) Hematopoietic cell clusters in the aortic region at
10.5 dpc. CD34 (red), Kit (green), and TOTO-3 (blue). (b) Hematopoietic cell clusters in the placenta at 10.5 dpc.
CD31 (red), c-Kit (green), and TOTO-3 (blue). (c) Hematopoietic progenitor cells in the liver at 12.5 dpc. DLK-1
(red), c-Kit (green), and TOTO-3 (blue). Although cryosections of 7–10 µm thickness have been widely used
previously for immunohistochemistry, sections shown here were of 20 µm and provide clear images of hema-
topoietic cell clusters and surrounding cells
3.2 Flow Cytometry Carry out all procedures at room temperature unless otherwise
specified.
1. Obtain placentas or fetal livers from pregnant mothers. Remove
deciduas and umbilical vessels from placentas.
2. Pass placentas or livers through 21-gauge needles to disrupt
tissue prior to collagenase treatment (see Note 6).
3. Put tissues into collagenase solution and incubate on a shaker
for 30 min at 37 °C (see Note 7). After incubation, pipette tis-
sue up and down very gently approximately 10 times to obtain
a single-cell suspension.
4. Filter suspension through 40-μm nylon cell strainers.
5. Add PBS to the suspension and centrifuge at 900 × g for 5 min.
6. For hemolysis, add lysing buffer to samples and wait for
15 min.
7. Centrifuge cells at 900 × g for 5 min and rinse with PBS.
Resuspend the cell pellet in 100–200 μl PBS.
8. Add all color-conjugated antibodies to the single-cell suspen-
sion. Add 0.3 μl of each antibody per 1.0 × 106 cells.
9. Incubate samples on ice for at least 30 min.
10. Add PI buffer to the suspension to remove dead cells by flow
cytometry.
11. Endothelial cells are defined as CD31+/CD34+/c-Kit−/
Ter119−/CD45−, mesenchymal cells as CD31−/CD34−/c-Kit−/
Ter119−/CD45−, hepatoblasts as CD45−/Ter119−/DLK-1+,
and sinusoid endothelial cells as CD45−/Ter119−/LYVE-1+/
c-Kit- c-Kit-Ter119-CD45-
Endothelial
cells
Ter119
SSC
CD31
10.5 dpc
AGM region
Mesenchymal
cells
c-Kit CD45 CD34
c-Kit- c-Kit-Ter119-CD45-
Endothelial
cells
SSC
Ter119
CD31
11.5 dpc
placenta
Mesenchymal
64.4 cells
c-Kit CD45 CD34
DLK-1-CD45-Ter119-
104 104
Sinusoid
103 103 2.17 Endothelial
CD45/Ter119
cells
12.5 dpc
CD31
102 102
fetal liver
1.52 101
101 Hepatoblasts
0 0
0 101 102 103 104 0 101 102 103 104
Dlk-1 Lyve-1
Fig. 2 Sorting of endothelial and mesenchymal cells by flow cytometry. Single-cell suspensions were prepared
from the aorta-gonad-mesonephros (AGM) region at 10.5 dpc (lower), placenta at 11.5 dpc (middle), and fetal
liver at 12.5 dpc (lower). Endothelial cells (CD31+/CD34+/Kit−/Ter119−/CD45−) and mesenchymal cells (CD31−/
CD34−/Kit−/Ter119−/CD45−) were sorted out from the AGM region and placenta, and hepatoblasts (CD45−/
Ter119−/DLK-1+) and sinusoid endothelial cells (CD45−/Ter119−/LYVE-1+/CD31+) were sorted from fetal liver
by flow cytometry
CD31+. Set the gate on flow cytometer for each population

and begin isolation (Fig. 2).
12. Collect isolated cells in RNA later for RNA extraction.
3.3 Laser Capture Carry out all procedures at 4 °C (see Note 8).
Microdissection
1. For this procedure, do not fix embryos and omit the sucrose
System equilibration step.
2. Embed placentas in OCT compound and freeze in liquid
nitrogen.
3. Slice tissues at 20 μm thickness with a cryostat and transfer to
glass slides. Place samples on ice and immediately store at
−80 °C until use.
c-Kit
Real-time PCR
LCM
Fig. 3 Laser capture microdissection (LCM) strategy. After staining placenta sections with anti-c-Kit antibody,
the edges of hematopoietic cell clusters expressing c-Kit and prospective niche cells surrounding those cells
are simultaneously marked under a microscope using software equipped with an LCM system. Hematopoietic
cell clusters expressing c-Kit (the center black rings) are dissected and removed and residual niche cells are
dissected out and collected into a tube for PCR analysis
4. After thawing, wash frozen sections in PBS 3 times for 1 min

each and block in 1 % BSA in PBS for 1 min. Incubate samples
with primary antibody solution for 60 min (see Note 9).
5. After washing 3 times in PBS, incubate sections with second-
ary antibody solution for 60 min to detect c-Kit-positive cells.
6. Dehydrate sections using an ethanol series (75, 90, 100 %),
each step for 30 s.
7. In this analysis, consider fluorescent c-Kit-positive cell aggre-
gates to be stem cell clusters. Mark and cut sections by laser
based on fluorescence. Remove c-Kit-positive cell aggregates
in placentas, and then capture niche cells (Fig. 3).
8. Transfer cells to microcentrifuge tubes containing 10 μl extrac-
tion buffer using a Pico Pure™ RNA Isolation Kit (see Note 10).
4 Notes
1. When considering the primary antibody combination, do not

choose antibodies raised in the same host species to avoid
cross-reactivity.
2. Collagenase should be stocked at −30 °C and added immedi-
ately before use.
3. Dry cryosections overnight at room temperature to minimize
the chance of detachment from the slide.
4. Incubation periods longer than 30 min result in high nonspe-
cific fluorescence in confocal analysis. Do not exceed this
period.
5. Slides stored at 4 °C in the dark retain fluorescence for approx-
imately 1 month.
6. When processing samples containing large tissue fragments, an

18- rather than a 21-gauge needle may be preferable. Chopping
samples with a razor blade before passage through the needle
can also be considered.
7. Treatment time is important. Do not exceed 30 min since cells
become damaged and an adequate number of cells may not be
obtained by following flow cytometry.
8. Since this method requires RNA extraction, the temperature
for procedures should be at 4 °C, and the time to RNA extrac-
tion should be shortened.
9. Apply solutions to slides very gently in order not to lose pla-
cental sections, as they are fragile and can become detached
from slides due to the absence of fixation.
10. Generally, the RNA yield extracted from a small number of
target cells captured by LCM is low. Therefore, we recom-
mend the use of RNA extraction kits known to enable the
highest recovery.
Acknowledgments
We thank The Ministry of Education, Culture, Sports, Science and

Technology; The Ministry of Health, Labor and Welfare; and The
Japan Society for the Promotion of Science for grant support, and
Dr. Elise Lamar for critical reading of the manuscript.
References
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Qualitative and quantitative aspects of hema- hematopoiesis is autonomously initiated by the
topoietic cell development in the mammalian AGM region. Cell 86:897–906
embryo. Immunol Today 19:228–236 8. Cumano A, Dieterlen-Lievre F, Godin I
2. McGrath KE, Palis J (2005) Hematopoiesis in (1996) Lymphoid potential, probed before
the yolk sac: more than meets the eye. Exp circulation in mouse, is restricted to caudal
Hematol 33:1021–1028 intraembryonic splanchnopleura. Cell 86:
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modern interpretations. Exp Hematol 33: Bodine DM, Orlic D (1997) Characterization
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4. Sugiyama D, Tsuji K (2006) Definitive hema- in the day 9 murine yolk sac. Immunity 7:
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6. Palis J, Robertson S, Kennedy M, Wall C, nopleures by aorta-gonad-mesonephros
Keller G (1999) Development of erythroid region-derived stromal cells. Blood 98:6–12
and myeloid progenitors in the yolk sac and 11. Sugiyama D, Ogawa M, Nakao K, Osumi N,
embryo proper of the mouse. Development Nishikawa S, Nishikawa S, Arai K, Nakahata T,
126:5073–5084 Tsuji K (2007) B cell potential can be obtained
from pre-circulatory yolk sac, but with low 18. Ema H, Nakauchi H (2000) Expansion of
frequency. Dev Biol 301:53–61 hematopoietic stem cells in the developing
12. Samokhvalov IM, Samokhvalova NI, liver of a mouse embryo. Blood 95:
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33:1048–1054 Hug C, Lodish HF (2006) Angiopoietin-like
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Chapter 7
Isolation and Enrichment of Stro-1 Immunoselected

Mesenchymal Stem Cells from Adult Human Bone Marrow
Emma L. Williams, Kate White, and Richard O.C. Oreffo
Abstract
The availability of mesenchymal stem cells (MSCs) or skeletal stem cells (SSCs) is vital to many of the tissue
engineering strategies currently being developed for repairing bone and cartilage. One difficulty with using
this cell population is that SSCs represent only a small fraction of the cells available from an individual patient’s
bone marrow sample, typically less than 1 in 10,000. Therefore, methods have been devised to enrich the
proportion of MSCs obtained from a bone marrow sample using hybridoma cell lines to generate antibodies
to cell surface antigens specific for MSCs. Stro-1 is the most widely targeted of these cell surface antigens. The
protocol described overleaf is used to isolate and enrich the Stro-1 positive fraction of cells from a bone mar-
row aspirate to provide a sample enriched for MSCs for use in both in vitro and in vivo studies.
Key words Stro-1, Mesenchymal stem cells, Skeletal stem cells, Bone marrow, Magnetic-activated cell
sorting
1 Introduction
Multipotent stem cells are a key component of any tissue engineering

strategy. For bone and cartilage, the key cell type is the skeletal
stem cell (SSC), commonly referred to as the mesenchymal stem
cell (MSC). MSCs have the potential to develop into any of the
tissues derived from the embryonic mesoderm, namely bone, carti-
lage, muscle, fat, and connective tissue, given appropriate conditions.
MSCs can either be isolated directly or potentially induced by
transfection (induced pluripotent stem cells—iPS cells). Bone mar-
row aspirates or peripheral blood are the most commonly used
sources of MSCs, although other sources such as adipose tissue are
now also available.
One of the difficulties with extracting MSCs from, for exam-
ple, bone marrow aspirates is that they represent only a small frac-
tion of the total cell population within the sample, typically less
67
68 Emma L. Williams et al.
Fig. 1 (a) Stro-1 expression by HK cells: Primary antibody—Stro-1 hybridoma supernatant (neat); secondary
antibody—Alexa Fluor 488 (green) goat-anti-mouse IgM (1:50 dilution); nuclei counterstained with DAPI (blue,
1:100 dilution). (b) Negative control showing positive nuclear staining with DAPI only
than 1 in 10,000. Although several markers of multipotency have

been identified, as yet there is no cell surface marker that is highly
specific and sensitive for identifying MSCs alone. This is crucial for
ensuring reproducibility of experiments performed using this cell
type. Several candidate markers have been suggested including
Stro-1, CD106, and CD73 [1]. Stro-1 is a monoclonal antibody
to a cell surface antigen expressed by a subpopulation of bone mar-
row stromal cells (BMSCs) (Fig. 1). The Stro-1 positive fraction
identified by this antibody has been previously shown to be signifi-
cantly enriched for colony-forming units-fibroblastic (CFU-Fs)
[2, 3]. Stro-1 is now used routinely for this purpose and further
research is in progress to determine whether combinations of
Stro-1 and some of the other markers currently available will
improve isolation of MSCs without compromising accuracy.
The protocol described below is used to isolate the Stro-1
positive fraction of cells from a bone marrow aspirate to provide
a sample enriched for MSCs for use in both in vitro and in vivo
studies. The BMSCs are immunolabelled with Stro-1 antibody
(mouse IgM), and then magnetically labelled with rat-anti-mouse
IgM microbeads. The Stro-1 positive fraction is then separated
from the unlabelled fraction by placing the sample within the
magnetic field of a MACS separator. This holds the magnetic
beads of the Stro-1 positive fraction within a central column,
whilst the negative fraction is washed through the column. Once
the separation column is removed from the magnetic field, the
Stro-1 positive fraction is obtained by washing the magnetic
beads through the column.
Stro-1 Based Isolation of MSCs from Adult Human Bone Marrow 69
2 Materials
2.1 Preparation of 1. Add 1 tub of powdered alpha Modified Eagle’s medium (Gibco,
Alpha-MEM Medium UK) to a clean 1 l volumetric flask using a spatula and funnel.
2. Add 1 l of distilled water, followed by 2.2 g of sodium hydro-
gen carbonate.
3. Affix stopper and invert to mix. Remove stopper and using a
magnetic “flea,” mix thoroughly on a bench top stirrer for
approximately 1 h.
4. Filter sterilize the medium into autoclaved glass bottles, using
a bottle-top filter (Nalgene) prior to use.
2.2 Preparation 1. Blocking buffer: 7 ml α-MEM (Gibco), 2 ml AB human serum

of Blocking Buffer (Sigma), 0.2 g bovine serum albumin (PAA), 1 ml fetal calf
serum (FCS) (Invitrogen).
2. Combine all of the above constituents and filter sterilize using
a 20 ml syringe and 0.2 μm syringe filter before use.
2.3 Preparation 1. For this step we use a hybridoma cell line, maintained within
of Stro-1 Hybridoma the lab, for which we keep frozen aliquots in liquid nitrogen.
Supernatant 2. Day 1 Wake cells up from liquid nitrogen by adding the con-
(See Notes 1–5) tents of one vial to a T75 containing 20 ml of DMEM
(PAA) + 20 % FCS.
3. Day 2/3 Check the cell growth—if healthy and at high density,
split into two T75s, adding 20 ml DMEM + 10 % FCS to each
flask.
4. Day 4/5 Check cells again—if very dense split again into two
T150s, adding 40–50 ml DMEM + 10 % FCS to each flask.
5. Day 6/7 Once the cells are growing well, switch to using
DMEM + 5 % FCS. Cells will continue to need splitting every
2–3 days to keep them healthy. Additional cells that are not
required for use can be frozen down in FCS + 10 % DMSO and
stored in liquid nitrogen.
6. Once the hybridoma cells have been growing well for about 2
weeks, pellet the cells from 1 confluent T150 and resuspend
the pellet in 10 ml DMEM + 20 % FCS. Split the remaining
T150 into 2× T150 and top up the medium to 50 ml per flask
with DMEM + 5 % FCS.
7. Wet the membrane of a CL flask (Integra Celline Classic 350
bioreactor I90010) by adding 15 ml of plain DMEM to the
medium compartment (green lid). Leave for 5 min.
8. Inoculate the cells suspended in DMEM + 20 % FCS into the
cell compartment (white lid). Remove all air bubbles by pipet-
ting the medium up and down and tipping flask. Replace the
cap tightly.
9. Add 200 ml plain DMEM to the medium compartment and

tightly fasten the lid.
10. After 2–3 days take 30 ml out of one of the T150s, spin down,
and suspend in DMEM + 20 % FCS. Remove 2 ml from the cell
compartment of the CL flask and mix in the new cells by pipet-
ting up and down, removing any bubbles. Top up the medium
in the T150 with DMEM + 5 % FCS. Keep freezing down sam-
ples from the T150s when confluent.
11. Leave the CL flask for a week before sampling but change the
medium compartment as required during this period.
12. Take 40 ml out of each T150, spin down, and pool in 10 ml
DMEM + 20 % FCS. Top the T150s back up to 50 ml with
DMEM + 5 % FCS.
13. Take 11 ml out of the cell compartment and replace with cells
from the T150s.
14. Spin down cells from the CL flask and keep the supernatant.
Store at −20 °C until required.
15. Remove 150 ml of medium from the medium compartment
and replace with fresh DMEM.
2.4 Preparation 1. MACS Buffer: 1 l phosphate-buffered saline (PBS), 5 g bovine

of MACS Buffer serum albumin (0.5 %), 0.74448 g EDTA (disodium salt) (2 mM).
2. Combine all of the above and filter sterilize using a 20 ml
syringe and 0.2 μm syringe filter before use. Store at 4 °C. The
bottle needs to be degassed using a vacuum pump prior to
each use for about 20 min.
3 Methods
3.1 Bone Marrow 1. Under sterile conditions within a Class II extraction hood,
Isolation transfer the bone marrow sample from the universal tube it was
placed in the operating theatre to a 50 ml Falcon tube (Greiner
Bio-One, UK).
2. Suspend the bone marrow sample in 10 ml of alpha modified
Eagle’s medium (α-MEM—Gibco, UK) and 1 % penicillin/
streptomycin (P/S—diluted from 100× concentrate, PAA,
UK) and shake vigorously.
3. Pipette the bone marrow suspension into a new Falcon tube.
Resuspend the remaining bone marrow sample in a further
10 ml α-MEM + P/S and transfer to the new Falcon tube,
repeating this until the bone fragments are white and clean.
4. Centrifuge the cell suspension at 250 × g for 4 min at room
temperature to obtain a cell pellet.
5. Carefully pour off the supernatant and resuspend the cell pellet
in 10 ml α-MEM + P/S, using the mechanical drawing action
of the pipette to break up the cell pellet.
6. Resuspend the cell pellet in 25 ml α-MEM + P/S and pipette it
through a cell strainer (0.70 μm pore size (Fisher)) into another
Falcon tube (see Note 6).
3.2 Removal of Red 1. Prepare the bone marrow sample as described above.
Blood Cells Using 2. Pipette 20 ml of lymphocyte separation medium (LSM) (PAA)
Lymphocyte into a fresh Falcon tube (see Note 7). Using a 3 ml pastette,
Separation Medium add the cell suspension very carefully onto the LSM, holding
the pastette close to the side of the tube and just below the
meniscus of the LSM to add the cell suspension as a steady
stream and form a uniform layer.
3. Centrifuge the LSM/cell suspension at 800 × g for 40 min at
18 °C, with the brakes off (see Note 8).
4. Remove the Falcon tube from the centrifuge. There should be
a layer of medium at the top, bone marrow mononuclear cells
between this and the LSM, and the blood cells at the bottom
of the tube.
5. Using a pastette, carefully remove the cell layer, followed by
the medium into a new Falcon tube and top up to 50 ml with
α-MEM + P/S.
6. Centrifuge twice at 240 × g for 10 min at 4 °C to remove all
traces of LSM.
3.3 MACS Stro-1 1. After the final centrifugation step of the mononuclear cell sep-
Isolation aration described above, pour off the supernatant and resus-
pend the pellet in 2 ml of blocking buffer (see Subheading 2.2).
2. Place in the fridge for 30 min.
3. Thaw the Stro-1 hybridoma supernatant and keep in the fridge
ready for use.
4. Remove the cell suspension from the fridge and add 8 ml of
chilled MACS buffer, to give a total volume of 10 ml (see
Subheading 2.4). Centrifuge at 300 × g for 5 min.
5. Pour off the supernatant and resuspend the cells in 1 ml undi-
luted Stro-1 hybridoma supernatant. Incubate in the fridge for
30 min, mixing regularly.
6. Centrifuge the cell suspension at 300 × g for 5 min. Pour off
the supernatant and wash the cells in 20 ml of chilled MACS
buffer, centrifuging at 300 × g for 5 min, for a total of three
washes.
7. Do a cell count and resuspend in 80 μl MACS buffer per 1 × 107
cells.
8. Add 20 μl of MACS rat anti-mouse IgM microbeads (Miltenyi

Biotech) to the cell suspension per 1 × 107 cells, mix, and incu-
bate in the fridge for 30 min.
9. Stain the cells with Stro-1 FITC antibody at 1 in 200 dilution
(5 μl FITC in 1 ml MACS buffer). Leave on for 15 min
(OPTIONAL STEP).
10. Wash cells three times with chilled MACS buffer. Resuspend
the cell pellet in 2 ml MACS buffer.
11. Place the magnetic column in its holder and push into the
bracket.
12. Label two 15 ml tubes, one negative (−ve) and one positive
(+ve) and place in a rack underneath the column.
13. Fill the column with 3 ml MACS buffer and let it run through.
14. Add the 2 ml cell suspension with a pastette.
15. Add 3 ml MACS buffer to wash out all the unsorted cells into
the −ve 15 ml tube. Repeat three times.
16. Remove the −ve tube. Take out the column and place in the
+ve tube. Add 5 ml fresh MACS buffer to the column. Place
the plunger at the top of the tube and press down firmly and
swiftly to push the positive cells out.
17. Wash the positive cells twice in α-MEM, centrifuging them at
300 × g for 5 min at 4 °C each time. Do another cell count
before resuspending the cells in α-MEM + 1 % penicillin/strep-
tomycin + 10 % FCS and seeding them into a T75. Incubate at
37 °C and 5 % CO2. Once cells reach 80–90 % confluence they
can then be passaged and placed in the required differentiation
medium for the planned experiment (e.g., basal and osteo-
genic medium).
4 Notes
1. When culturing Stro hybridoma cells, check the cells every day
to assess growth. Timescales given are approximate and have
to be adjusted according to how the cells look.
2. Avoid spinning down Stro hybridoma cells as much as possible
as they do not tolerate this very well.
3. Do not use penicillin/streptomycin in the cell cultures.
4. Healthy Stro hybridoma cells look like round shiny spheres
and grow well at high density.
5. The color of the medium is a good indicator of cell growth
(red—good, orange—need splitting, yellow—dead cells).
6. When pipetting the cell suspension through the cell strainer,
gently scrape the bottom of the filter to prevent it from clog-
ging up.
7. LSM must be kept at room temperature and wrapped in foil to

protect it from the light.
8. It is crucial to spin the LSM with the brakes off in order to
allow the discrete cell layers to form. This will take about an
hour.
References
1. Kolf CM, Cho E, Tuan RS (2007) Mesenchymal antibody, STRO-1. Blood 78(1):55–62, Epub
stromal cells. Biology of adult mesenchymal 1991/07/01
stem cells: regulation of niche, self-renewal and 3. Stewart K, Walsh S, Screen J, Jefferiss CM,
differentiation. Arthritis Res Ther 9(1):204, Chainey J, Jordan GR et al (1999) Further char-
Epub 2007/02/24 acterization of cells expressing STRO-1 in cul-
2. Simmons PJ, Torok-Storb B (1991) tures of adult human bone marrow stromal cells.
Identification of stromal cell precursors in J Bone Miner Res 14(8):1345–1356, Epub
human bone marrow by a novel monoclonal 1999/08/24
Chapter 8
Primary Marrow-Derived Stromal Cells:

Isolation and Manipulation
Aravind Ramakrishnan, Beverly Torok-Storb, and Manoj M. Pillai
Abstract
Marrow stromal cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in
aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give
rise to several mesenchymal lineages. “MSC” also refers to mesenchymal stem cells which implies that all
cells in the population are multipotent. It is generally agreed that while there may be a few multipotent
stem cells in an MSC population the majority are not stem cells. In either case MSCs do not produce
hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone mar-
row is their most common source for research and clinical use. Primary MSC populations can be derived
from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular het-
erogeneity within a culture and how this may vary from donor to donor. In this chapter, we describe
methodology to derive primary MSCs from bone marrow screens, an otherwise discarded by-product of
bone marrow harvests used for clinical transplantation. We also describe some useful techniques to charac-
terize and manipulate MSCs—both primary and immortalized cell lines.
Key words Marrow stromal cells (MSCs), Bone marrow screen, Reverse-transfection, FACS,
AutoMACS, CD146, siRNA, miRNA, Long-term culture (LTC)
1 Introduction
1.1 Stromal Cells Maintenance of normal hematopoiesis at all stages of ontogeny

as an Integral requires a complementary microenvironment (ME), which in adult
Component of the vertebrates resides within the bone marrow [1, 2]. Early evidence
Microenvironment (ME) for the importance of the ME came from experiments in naturally
occurring mutant mice. The compound heterozygote SL/SLd
mouse could be rescued from effects of low-dose radiation by the
transplantation of a wild-type spleen but not wild-type hematopoi-
etic cells suggesting that the defect lies in the ME (the soil) rather than
the hematopoietic stem/progenitor cell (HSPC, or the seed) [3].
The SL locus was subsequently discovered to encode for Kit ligand
(KITL or stem cell factor, SCF), a cytokine that is produced by
the ME with nonredundant regulatory functions for HSPC
75
76 Aravind Ramakrishnan et al.
maintenance [4, 5]. Despite early enthusiasm that KITL might be

the critical ME-derived gene product that defines the hematopoietic
ME, it has since then become abundantly clear that hematopoietic
regulation by the ME is enormously complex with contribution
of several cell types and dozens of secreted or surface-bound
factors [6]. Some of these factors such as CXCL12, SCF, and
ANGPT1 have nonredundant functions while others such as the
Notch and Wnt ligands are redundant, as evidenced by murine
gene knockout models [7–14]. These factors are typically not
restricted in origin to a single cell type within the ME, further
complicating attempts to precisely define the cellular and anatomi-
cal location of specific functional niches within the ME [6, 15, 16].
It is now generally accepted that cells of mesenchymal origin
such as the osteoblast, endothelium, fibroblast-like stromal cells,
and adipocytes as well as cells of hematopoietic origin such as
macrophages, osteoclasts, and megakaryocytes functionally con-
tribute to the regulation of the HSPC and its subsequent progeny
within the ME. The terms “stroma” or “stromal cells” have been
historically used to denote the fibroblast-like cells of mesenchymal
origin found in primary bone marrow long-term cultures (LTCs as
detailed later). Precise demarcation of stroma vs. other cells of
mesenchymal origin (such as osteoblasts) is problematic with
immune-phenotypic techniques given overlap of surface markers
and incomplete understanding of different stages of their differen-
tiation from a putative common precursor in vivo. While fibroblast-
like stromal cells are best appreciated in in vitro cultures where
they proliferate luxuriantly in serum-rich medium to form adher-
ent layers, they are more difficult to define in vivo due to (1) their
much smaller numerical proportion in comparison to the rapidly
proliferating hematopoietic cells and (2) their thin and pleomor-
phic morphology that renders direct visualization of cells in bone
marrow sections challenging but not impossible with specific stains.
Consequently, most studies of stromal cells until recently have
been in the in vitro system. Use of genetically modified mouse
models using tissue-specific promoters (such as osterix and nestin
promoters) and surface markers such as CD146 (in human primary
samples) has been reported in the past few years and has signifi-
cantly accelerated our understanding of stromal cells and their
function in vivo [17–19].
1.2 MSC: Misleading Most of the initial interest in these cells after their initial descrip-
Misnomer tion by Dexter centered around the mechanistic basis of their
interaction between hematopoietic cells and how they support
hematopoiesis [1, 20]. The mid-1990s, however, witnessed a sig-
nificant interest in stromal cells, which came to be denoted inap-
propriately as mesenchymal stem cells (MSCs), for a wide spectrum
of clinical uses ranging from regeneration of damaged tissues like
heart and liver to immune modulation of allogeneic graft versus
Primary Marrow-Derived Stromal Cells: Isolation and Manipulation 77
host disease [21–24]. Most of these anecdotal observations failed

to translate to tangible benefits in larger trials which has dampened
enthusiasm of this mode of cellular therapy. Nevertheless some
investigators are committed to understanding how infusion of cul-
tured stromal cells, which usually are completely mismatched to
the recipient, and rapidly filtered out of systemic circulation by the
lungs, could influence tissue regeneration or allo-immune reactions
in select clinical situations [25–28]. In 2001, a report described the
existence of rare multipotent adult progenitor cells (MAPCs) with
embryonic stem cell-like potential in MSC populations [29–31].
The MAPC had the ability to “transdifferentiate” into multiple tis-
sues but also to revert back to embryonic potential and contribute
to all three germ layers. This obviously caused frenzied excitement
as the existence of MAPC could eliminate the need for harvesting
blastocysts. The existence of MAPCs has since then been largely
disavowed and there is general consensus that stromal precursors
are not capable of differentiation to tissues outside traditionally rec-
ognized mesenchymal lineages, unless deliberately reprogrammed
by the forced expression of embryonic transcription factors [32–37].
A comprehensive review of the history and controversies surround-
ing clinical applications of MSCs and MAPCs is outside the scope
of this manuscript and the reader is referred to several papers for
further details [25–27, 29, 31, 32, 36, 38].
1.2.1 Defining MSC MSCs are typically defined as cells that possess the ability to form
colony-forming unit-fibroblast (CFU-F) and differentiate into
multiple mesenchymal cell types (including osteoblasts, chondro-
cytes, adipocytes, and bone marrow stromal cells) under appropri-
ate in vitro culture conditions [39, 40]. Although it is generally
accepted that these mesenchymal lineages likely originate from
common precursors (much in the same way that a single self-
renewing hematopoietic stem cell can self-renew and give rise to
mature hematopoietic lineages), unequivocal evidence for such a
single common precursor that repopulate all mesenchymal lineages
has been lacking [38]. This is due to both the obvious difficulty in
depleting all mesenchymal cells from a host and the difficulty in
reliably transplanting a single stromal cell into such a depleted
host. Although only a small portion of the stromal cells from fresh
bone marrow isolates or early bone marrow cultures would be con-
sidered true MSCs, the term is often inappropriately applied to all
cells in a marrow-derived stromal culture.
Given the heterogeneity of the stromal population, several
groups have attempted to enrich for the CFU-F population using
cell surface markers. The first effort reported was the generation of
the murine monoclonal antibody STRO-1, an IgM antibody spe-
cific for an undefined polysaccharide motif. STRO-1 binds to
approximately 10 % of bone marrow mononuclear cells (BMMNCs),
most of which are nucleated erythroid cells [41]. When nucleated
erythroids are excluded by glycophorin A positivity, STRO-1-

positive cells define a population that is 100-fold enriched for
CFU-F. Furthermore, STRO-1 can be easily identified in the stro-
mal cell layers of the LTC. Despite widespread use of this antibody
to enrich for CFU-Fs, an obvious limitation is its presence on
hematopoietic cells, preventing use in bone marrow sections or
mixed primary LTCs. Over the past two decades, several groups
have tried to extend on this work by attempting to isolate MSC
using various cell surface markers including CD49a, CD63, CD90,
NGFR, CD105, CD106, CD140b, and CD146 [17, 42–47]. Of
these markers, only cells that express high levels of CD146 have
been shown to transfer a functional hematopoietic ME when trans-
planted into immunodeficient mice [17]. This antibody can also be
used to identify progenitors in vivo by immune histochemistry (see
figure) and our group has found this to be a useful marker to enrich
for MSC (details of protocol explained later) [48].
1.3 MSC Cultures Although culture conditions can be varied to promote stromal
growth over hematopoietic growth resulting in further enrichment
of fibroblast-like cells, they nevertheless remain highly heteroge-
neous and contaminated with cells of hematopoietic origin, most
commonly macrophages. These cultures are typically referred to as
MSC cultures. Setting up primary MSC cultures is relatively
straightforward, but one must expect wide variation among donors
and between cultures from the same donor. Differences in growth
kinetics and relative proportions of different cell types are com-
mon, as is the ability to support different hematopoietic stages and
lineages. MSC quality is also influenced by viral infections like
cytomegaloviruses (CMV), and technical issues which are often
unavoidable, including a delay in processing, or a significant con-
tamination by erythroid cells necessitating hypotonic hemolysis
of samples [49]. Even adjusting for these factors, the phenotypic
and functional characteristics of these cells are difficult to predict.
Cloned stromal cell lines have circumvented many of these
problems; however, results from cell lines must be reproduced in
primary cells to be considered valid. While cell lines are readily
available, descriptions of current techniques to isolate primary
cells and manipulate them in vitro are not as available.
Most tissues that have discernible connective tissue components
have mesenchymal progenitors that can be grown in cultures that
resemble MSC [38]. Bone marrow has remained the most com-
monly used and best studied source of MSCs due to relative ease of
access by bone marrow aspiration. Although normal bone marrow
aspirates can be obtained by volunteer donations or remunerated
study participants, this is often problematic for obvious reasons.
Although relatively safe, bone marrow aspirates are painful and inva-
sive and major complications like severe hemorrhage from a severed
aberrant blood vessel or damage to internal organs have occurred.
An alternate strategy that our group and others has successfully

used for several years is to salvage cells from bone marrow screens
used to prepare marrow for clinical transplantation. The shift away
from BMMNCs to peripheral blood mononuclear cells (PBMC) as
the preferred source of stem cells for clinical use has meant that the
number of bone marrow harvests has reduced drastically in the
past two decades. Bone marrow harvests however continue to be
performed for specific indications (like pediatric donors, for donor
conditions like sickle cell trait, or as part of study protocols that
require bone marrow as a source for the stem cells) and hence
result in a respectable number of marrow screens available for
extraction of BMMNCs in most larger centers. The methods
described in this manuscript focus on the use of bone marrow
screens for MSCs, but can obviously be used for BMMNCs from a
fresh marrow aspirate should that be available.
2 Materials
2.1 IRB Approval As with any other work involving biological materials, appropriate
and Personnel Training training of personnel, and up-to-date institutional approval of
protocols, is essential. Institutional Review Board (IRB) approval
has to be in place before the work commences. Depending on the
particular institution, the investigators may be eligible for expedited
review and human subject exclusion given that the screens may be
provided after removing personal identifiers and that the material is
otherwise discarded. Proper training and certification of personnel
in handling of biohazardous materials are similarly critical given that
the marrow screens could be a source of infection although donors
are typically tested extensively prior to marrow harvest (see Note 1).
2.2 Cell and Tissue Proper cell and tissue culture techniques and equipment are of
Culture Setup critical importance in any tissue culture methodology, but espe-
cially so for primary MSC cultures given that they could be con-
taminated at multiple steps of their setup and maintenance, and the
relatively long periods (weeks to months) that they may need to be
maintained. Most laboratories which perform tissue culture are set
up with biosafety laminar flow cabinets (or TC hoods), incubators
that can maintain temperature, CO2 content and humidity (TC
incubator), benchtop centrifuges to spin down cells, vacuum traps,
water baths, standard inverted microscopes (for cell counting and
monitoring of cell growth), etc. The setup and maintenance of
these are hence not discussed in detail here.
2.3 Bone Bone marrow screens are filters used to remove particulate materials
Marrow Screens including marrow spicules, fat globules, and small clots from the
bone marrow aspirated from donors for the purpose of transplanta-
tion (Fig. 1). They can be procured from the cell processing or
Fig. 1 Schematic of bone marrow harvest and screens. The bone marrow aspi-
rate collected from donors is hooked up typically to two filters called screens
connected in series (500 μM first followed by 200 μM) to filter out particulate
matter including spicules, clots, and tissue fragments. The filtrate is collected for
infusion (processed marrow). The marrow screens are then made available to
research laboratories with the entry port and exit connected to each other
apheresis laboratories of clinical centers where bone marrow aspirates

are processed and can be provided without any patient identifiers
other than age, sex, and other important pertinent clinical parame-
ters like CMV infection status. If personal identifying information is
excluded, this will typically allow for the samples to be considered
exempt from human subject research allowing for less stringent IRB
protocols. Given that the screens are clinical grade products, they
are typically sterile and contamination of their contents is unlikely at
the time of processing in the clinical laboratory. General aseptic
Fig. 2 Extraction of bone marrow mononuclear cells (BMMNC) from marrow screens. (a) Screens are typically
provided with the two filters (500 and 200 μM) connected in series as a sterile loop. (b) Carefully disengage
the entrance port of the 500 μM filter from the exit port of the 200 μM filter. (c) Pour any liquid contents left in
the screen to a 50 cc conical tube. (d) Inject 25 cc of PBS–EDTA into the entrance port of the 500 μM filter and
collect the run through carefully. (e) Try to dislodge any particulate material in the filters into the PBS–EDTA
solution. (f) Approximately 50 cc of bone marrow particulate material is eluted into a 50 cc conical tube.
(g) The cell pellet after the first wash is rich with erythroid cells which need to be mostly removed before
plating in culture. (h) Hemolysis of collected cells in hemolysis buffer at 37 °C. (i) Post-hemolysis and a further
wash in PBS–EDTA, cell pellet is pale and has little visible erythroid contamination
precautions at the time of transfer to the research laboratory would

suffice. Typically two screens of different pore size (500 and
200 μm) are serially connected to filter the marrow. These can be
attached to each other to form a sterile loop at the time of transfer
avoiding spillage of contents and contamination (Fig. 2a) attached
to each other. If your laboratory is in the process of setting up a
working relationship with the clinical cell processing laboratory for
purposes of procuring these screens, it would be worthwhile to
explain in detail to the facility supervisor and the staff the scientific
basis of your studies and the practical details of what you intend
to do with the screens as this will allow for expeditious transfer
(see Note 2).
2.4 Human Stromal Various groups have reported on immortalized stromal cell lines,
Cell Lines typically generated using retroviral vectors. Our group has previ-
ously reported on the use of human stromal cell lines isolated after
immortalization with retrovirally expressed human papilloma virus
(HPV) E6/E7 proteins; two cell lines termed HS5 and HS27a
have been used to represent functionally distinct stromal niches
that exist within the larger context of the adult vertebrate marrow
microenvironment [50]. HS5 represents a stromal phenotype that
secretes large quantities of cytokines, thus driving differentiation of
HSPC to mature myeloid lineages. HS27a in contrast produces
high levels of HSPC niche-associated genes (such as CXCL12,
Jagged1, and Angiopoietin1) that help maintain HSPC in their
primitive undifferentiated states (see Note 3).
2.5 Flow-Cytometry Flow-based sorting as described here for CD146 expression is typi-
and Sorting of Stromal cally available in large research centers where well-trained staffs per-
Cells Based on CD146 form the sorting using shared instruments (see Note 4). For magnetic
Expression: Flow bead sorting for enrichment of stromal precursors by removal of
Sorters and MACS CD14- and CD45-positive cells, we have utilized proprietary
Sorters magnet-conjugated antibodies from Miltenyi Biotec (see Note 5).
2.6 Medium for MSC Human MSCs can be grown in a variety of medium formulations
Growth and as long as they are replete with serum. These include alpha minimal
LTC Growth essential medium, Dulbecco’s modified Eagle’s medium (DMEM),
and RPMI-1640. DMEM commonly comes in low-glucose or
high-glucose variants and low-glucose formulations are routinely
used by most groups for propagating MSC. Although the other
base medium formulations have differences in components, in our
anecdotal experience, they can be used interchangeably as long as
they are serum supplemented. Fetal bovine serum (FBS) supple-
mented at 10–20 % final volume is considered essential for good
proliferation of all fibroblast cultures including bone marrow-
derived stromal cells. FBS is known to have high concentrations of
several fibroblast mitogens (from the PDGF and FGF family) (see
Note 6). Serum-free formulations with defined chemical additives
such as cytokines and small molecules, as have been developed for
hematopoietic colony growth, would be an important technical
advancement for the field; however at this time FBS remains a
necessary supplement for MSC cultures. Addition of antibiotics
such as penicillin–streptomycin and antifungal agents such as
amphotericin B should be considered, the latter especially in cli-
mates where mold contamination is known to occur in cultures
that have to be maintained for several weeks.
Medium for primary LTC is typically composed of Iscove’s
medium supplemented with screened horse serum (HS) in addition
to FBS and several other additives (see Note 7). Although the human
stromal cell lines Hs27a and Hs5 were initiated in LTC medium
they can be grown in RPMI-1640 supplemented with 10 % FBS.
2.7 Medium Thaw frozen FBS/HS at 4 °C overnight. Heat inactivate FBS/HS

Formulations by incubating at 56 °C for 30 min to inactivate complement. Filter
and Other Solutions sterilize using 0.45 μ bottle filter (make sure to agitate the serum
while in the filter chamber to prevent clogging of filter pores by
2.7.1 Fetal Bovine Serum
clots). Dispense into appropriate size (50 ml conicals or 100 mm
and Horse Serum
bottles) working aliquots and freeze at −20 °C.
2.7.2 Bovine Serum Dissolve 100 g fraction V BSA in 1 l of deionized water. Filter
Albumin, 10 % Solution sterilize using a low-protein-binding 0.45-μm filter. Store in
50–100 ml aliquots at −20 °C.
2.7.3 MSC Medium 50 ml of prescreened FBS.

(DMEM Based; 5 ml of 100× Penicillin/streptomycin.
Components Purchased
5 ml of 100× Amphotericin B (optional).
from Standard Suppliers as
GIBCO or Fisher Scientific) DMEM (low glucose, replete with pyruvate and glutamate) to a
total of 500 ml.
Mix components, filter sterilize with 0.45 μM bottle filter, and
store at 4 °C for use in less than 3 months.
2.7.4 LTC Medium 62.5 ml prescreened FBS (screened to support LTC growth) to a
final concentration of 12.5 %.
62.5 ml prescreened HS (screened to support LTC growth) to a
final concentration of 12.5 %.
5 ml of 100× Penicillin–streptomycin.
5 ml of 100× Amphotericin B (optional).
5 ml of L-glutamine.
5 ml of Pyruvate.
250 ml of 2× Isocove’s modified Dulbecco’s medium (IMDM)
prepared from powder (GIBCO).
500 μl of 103 M hydrocortisone stock (final concentration 106 M).
Distilled tissue culture-grade water (endotoxin free)—add to total
of 500 ml.
Mix all components and filter sterilize with 0.45 μm filter and
store at 4 °C for up to 3 months. They can also be frozen at −20 °C
for future use if larger volumes are made.
2.7.5 Stromal Cell Line 50 ml of prescreened FBS.

Medium (HS5 and HS27a) 5 ml of 100× Penicillin/streptomycin.
445 ml of RPMI-1640 (replete with pyruvate and glutamate).
Mix components, filter sterilize with 0.45 μm bottle filter, and
store at 4 °C for use in less than 3 months.
2.7.6 Solutions
10× PBS
and Buffers
NaCl 800 g
KCl 20 g
Na2HPO4 ⋅2H2O 144 g
KH2PO4 24 g
Dissolve in 10 l of deionized water, filter sterilize, and store at

4 °C. The pH is approximately 6.8 at this time. To make working
1× solution, take 100 ml of 10× stock and add 900 ml of deionized
water. The pH should adjust to 7.4 with dilution and this should
be verified with pH meter.
10× Hanks’ balanced salt solution (HBSS)

NaCl 80 g
KCl 4g
Na2HPO4·7H2O 0.9 g
KH2PO4 0.6 g
Add deionized water to 1 l and filter sterilize. Store at 4 °C.

To make 1× working stock, mix 100 ml of 10× stock with
800 ml of deionized water. Add 0.35 g NaHCO3 and 1 g of D-
GLUCOSE. Bring volume up to 1 l and adjust pH to 7.4. Store at 4 °C.
2.7.7 EDTA, 0.5 M Dissolve 186.1 g disodium ethylenediaminetetraacetic acid

(pH 8.0) (EDTA) dihydrate in 700 ml water.
Adjust pH to 8.0 with 10 M NaOH (~50 ml; add slowly).
Add water to 1 l and autoclave. To make 2 mm working stock, add
1 ml to of 0.5 M EDTA to 250 ml of PBS.
2.7.8 AutoMACS/FACS To 473 ml of 1× PBS, add 2 ml of 0.5 M EDTA, and 25 ml 10 %

Buffer (PBS with 2 mM BSA solution.
EDTA, 0.5% BSA)
2.7.9 PBS–EDTA To 500 ml 1× PBS, add 2 ml of 0.5 M EDTA.
2.7.10 Citrate Saline 50.3 g of KCl.

Solution (For Nonenzymatic 22.06 g of sodium citrate.
Cellular Dissociation)
Dissolve in tissue culture-grade water to total of 500 ml (for a 10×
solution). Filter sterilize or autoclave and store at 4 °C.
Make 1× solution (final concentration of KCl is 135 mM and

sodium citrate is 15 mM) as needed for use with tissue culture-
grade water.
Various commercial nonenzymatic preparations (from vendors
such as Millipore and Sigma-Aldrich) are also available.
2.7.11 Hemolytic Buffer

10× stock solution (1,000 ml)
NH4Cl 83.0 g
NaHCO3 10.0 g
EDTA 0.4 g
Dissolve salts in approximately 500 ml sterile water and after

all the salts have dissolved, make up the volume to a total of
1,000 ml. Filter sterilize with 0.45 μm filter and store at 4 °C.
Reconstitute 1× solution (typically not more than 50 ml at one
time) as needed.
2.7.12 Other Solutions Trypsin–EDTA (0.25 % or 0.05 % trypsin and 1 mM EDTA) avail-
and Buffers able from various vendors.
Phosphate-buffered saline (PBS).
PBS–EDTA (addition of EDTA to 2 mM final concentration).
Ficoll solution (1.073 or 1.077 g/ml) from various vendors.
2.7.13 Antibodies CD45 microbeads, Miltenyi Biotech, catalog #130-045-801.

Isotype: Mouse IgG2a.
CD14 microbeads, Miltenyi Biotech, catalog #130-050-201.
Isotype: Mouse IgG2a.
CD146 microbeads, Miltenyi Biotech, catalog #130-093-596.
Isotype: Mouse IgG1.
FITC Mouse anti-Human CD146, BDBiosciences, catalog #
560846.
Clone: P1H12, Isotype: Mouse IgG1, κ (same clone is also avail-
able from other vendors including eBiosciences).
2.7.14 Reverse OptiMEM Medium (Life/GIBCO catalogue # 31985–070).

Transfection Reagents Lipofectamine 2000 Reagent (Life/GIBCO catalogue #
11668019).
RPMI-1640 with 10 % FBS without antibiotics.
3 Methods
3.1 Isolating Bone As mentioned above, it is absolutely critical to maintain a sterile

Marrow Cells from working environment and minimize contamination of the cell prep-
Marrow Screens aration at each step of this protocol given that the cultures need to
be maintained for several weeks in nutrient-rich medium and hence
antibiotics/antifungal supplementation of culture medium alone
will be insufficient to prevent contamination by microbes.
1. Aliquot approximately 50 cc of PBS with EDTA in a 50 ml
conical tube.
2. Using a 18 gauge needle connected to a 50 cc syringe, aspirate
the PBS–EDTA to the syringe. Keep by the side for later use.
3. Spray down the screen with 70 % ethanol in the TC hood and
as mentioned above, screens are provided typically attached to
each other as a loop (Fig. 2a); this helps to keep the contents
sterile and prevent spillage of contents. Disengage the screens
from each other by opening the connectors (Fig. 2b) taking
care not to spill the contents of the screen. Carefully open the
connector such that the liquid contents can be poured out of
the inlet of the larger pore sized filter into a 50 cc conical tube
(for example, if 500 and 200 μm filters are connected in series,
pour out of the inlet of the 500 μm filter, Fig. 2c). This way the
larger trapped particles of bone spicules could be harvested.
4. Attach the 50 cc syringe directly to the connector of the screens
or through the 18 gauge and inject 25 ml of PBS–EDTA tak-
ing extreme care not to let the PBS–marrow mix to spill out.
Collect the flow through in the 50 cc conical tube (Fig. 2d, e).
Alternatively, one can reconnect the two screens and swish the
contents to dislodge of the filters into the PBS–EDTA solution
(for about 1–2 min) and pour the contents into the 50 cc coni-
cal tube.
5. Repeat the wash process (step 4) once for a total of about
50 cc washout (Fig. 2f).
6. Centrifuge the washout in an appropriate tabletop centrifuge
for 5 min at 4 °C to obtain a cell pellet (Fig. 2g). Carefully
aspirate the supernatant and immediately resuspend in either
PBS–EDTA or hemolytic buffer (to remove erythroid cells as
described in Subheadings 3.2 or 3.3) (see Note 8). Further pro-
cessing of the cell pellets can be done by either direct hemolysis
or density gradient separation. Getting rid of the erythroid cells
is necessary as lysis of erythroid cells in culture is known to
inhibit adequate stromal growth. In our experience, direct
hemolysis is usually sufficient for isolation of bone marrow cells
for MSC/long-term cultures since the hemolyzed sample can
be directly plated to culture without further delay. If the iso-
lated bone marrow cells are to be used for other purposes such
as flow sorting or there is a delay in further use of the cells, the

polymorphonuclear cells (PMNs or mature neutrophils) could
cause clumping of cells. Both protocols are hence described
below.
3.2 Hemolysis of 1. Add approximately 5 ml of 1× hemolytic buffer to the bone mar-

Bone Marrow Washout row pellet after the pellet is loosened by gentle “racking” (run-
ning across a plastic rack used for holding 15 cc conical tubes).
2. Incubate in a 37 °C water bath. Gently agitate every 30 s by
swirling the contents of the tube monitoring for a change in
color of the contents from “blood red” to “wine red” and rela-
tive clearing of contents (Fig. 2h). Limit total hemolysis time
to not more than 5 min to avoid cell clumping and lysis of
non-erythroid cells.
3. Bring tube back to the TC hood, and add 45 cc of PBS–EDTA
to the tube to make the volume up to 50 cc. Spin at 400 × g
force for 5 min.
4. Examine the tube contents for the adequacy of hemolysis
(both supernatant and cell pellet) (see Note 9). The final pellet
after adequate hemolysis would be pale and devoid of frank
erythroid presence (Fig. 2I).
3.3 Ficoll Density Density gradient separation based on the polysaccharide Ficoll to
Gradient Separation to separate whole blood or bone marrow to different components is
Yield BONE MARROW a useful technique for isolation of MSCs and stromal precursors
Mononuclear Cells since they are contained in the mononuclear fraction at the inter-
phase between Ficoll and diluted bone marrow. Layering of blood
or marrow on Ficoll is a delicate technique and it is recommended
that it is learned from those proficient with the technique and prac-
ticed on more easily obtainable samples like peripheral blood
before attempting on limited resources like bone marrow aspirate.
Ficoll density (g/cm3) 1.077 is classically used for BMMNC prepa-
ration from human bone marrow (see Note 10). Also important to
remember is that the gradient separation is not absolute and does
result in the loss of BMMNC (including the stromal precursors) to
other fractions. Hence if the sample is limited (such as part of a
clinical procurement) and to be used for direct plating for MSC
cultures, it is recommended that one consider direct hemolysis
instead of Ficoll gradient separation.
1. Move Ficoll from 4° fridge to TC hood at least 30 min before
separation since Ficoll density is sensitive to temperature. Do
not warm in a 37 °C water bath. To expedite equilibration to
room temperature, one can aliquot the Ficoll to the Falcon
tubes about 30 min before separation (step 3 of this protocol).
2. Dilute bone marrow with three volumes of medium HBSS.
Diluting the blood or the BM increases the yield of mononu-
clear cells and decreases the hang-up of RBCs.
Fig. 3 Extraction of bone marrow mononuclear cells (BMMNC) by Ficoll gradient separation. (a) 3 ml of Ficoll
at room temperature is aliquoted into 15 cc Falcon tubes. (b–d) 6–8 mf of dilute bone marrow is carefully
layered on the Ficoll layer taking care not to mix the two layers up. (e) After centrifugation at 400 × g for 30 min
at room temperature, different layers become apparent. The mononuclear cells are trapped in the layer
between Ficoll and serum. (f–j) With a sterile glass pipetted (stuffed with cotton at proximal end for seal), the
mononuclear layer is carefully collected minimizing suction of both the Ficoll layer between and the serum
layer above
3. Place 3.0 ml Ficoll into each clear 15 ml round-bottom tubes

(Falcon 2057). As noted above, it is critical to have Ficoll at room
temperature (RT) since the density is temperature dependent.
4. Tilt the tube to wet the side with Ficoll and then gently layer
6–8 ml of the diluted bone marrow on top (Fig. 3a–d). Pre-
wetting the side of the tube helps the bone marrow flow
smoothly down the side and prevents breaking the interface.
5. Centrifuge tubes at 400 × g force for 30 min at RT. Mononuclear

cells will appear as a white cloud at the interface and red cells
will be pelleted at the bottom of the tube (Fig. 3e).
6. Collect the interface (mononuclear cells) on top of the Ficoll
with a cotton-stuffed Pasteur pipette (Fig. 3f–j). Single-use
sterile plastic pipettes could also be used. Pool the interface
cells from two layers into a labeled 15 ml V tube, fill tube up
with HBSS to dilute the Ficoll, and wash it off the cells by
centrifuging at 400 × g force for 10 min, RT, to pellet cells (this
is the first wash).
7. Aspirate off supernatant and resuspend the pellet by tapping
the bottom of the tube, or by gently dragging the tube across
the top of a test tube rack (“racking”). Wash the cells with
10–15 ml HBSS.
8. If there is visible RBC contamination of the pellet, lyse the
RBCs by following the protocol as detailed in Subheading 3.2.
9. Then fill up tube with HBSS and centrifuge tube at 1,200 rpm
(400g), for 5 min. This slow spin keeps platelets from pelleting.
10. Consolidate cells to a single 15 ml tube, get a cell count, and
wash again as in step 8.
11. Cells can now be counted prior to plating in culture.
3.4 Setting Up Primary LTCs (or Dexter cultures) as originally described by

Human Marrow Dexter in 1977 is a marrow-derived primary culture that supports
Long-Term Cultures both hematopoietic and stromal growth and has been used as an in
vitro approximation of the complex cellular interactions in vivo for
decades [20]. The culture medium is hence optimized for hemato-
poietic growth in addition to stromal growth. In the course of days
to several weeks after setting up LTCs, stromal elements become
confluent and sustain primitive hematopoietic precursors within
stromal layers (seen as stacked phase-dark cells in phase contrast
microscopy and are referred to as cobblestone areas or CSAs (long
arrows in Fig. 4a, b)). As these progenitors divide and mature, they
are released to the medium, seen as phase-light cells (short arrows
in Fig. 4a, b). The cultures can be sustained by “demi-depletion”
by which half the volume of medium is removed every week or so
allowing to remove floating mature hematopoietic precursors,
replenishing nutrients while not completely depleting the cytokine
milieu created by the stromal cells. In contrast to MSC cultures,
LTCs are typically not split at confluence since the complex cellular
interactions between hematopoietic and stromal elements would
be perturbed and not easily reconstituted in the next passage.
1. Prepare BMMNC with techniques described in Subheadings 3.2
(typically adequate) or 3.3.
2. Count cells and set up in TC flasks/plates in LTC medium as
per the following scheme:
Fig. 4 Primary long-term cultures (LTCs or Dexter cultures). (a) Cartoon depicting
vertical layer of an LTC with stromal and hematopoietic elements. (b) Phase con-
trast micrograph of an LTC. Long arrows in both panels depict the cobblestone
areas (CSAs) comprising primitive hematopoietic precursors trapped within the
stromal layers and appear as “phase-dark” cells resembling a cobblestone.
Mature myeloid cells are released into the supernatant when the more primitive
precursor cells in the CSAs divide and mature (short arrow in both panels ) and
appear as “phase-light cells” in phase contrast micrographs
T75 Flask (or 100 mm dish) 15–20 × 106 cells in 12 ml

T25 Flask 5–10 × 106 cells in 5 ml
6-well plate 3 × 106 cells in 2–3 ml/well
24-well plate 1 × 106 in 1 ml/well
3. Culture cells at 37 °C, 5 % CO2, feeding every 7 days by demi-

depleting the medium (removing about half of the spent
medium which contains non-adherent monocytes and myeloid
cells) and replacing with equal volume of fresh LTC medium.
A confluent culture of fibroblasts, macrophages, and adipo-
cytes should be formed in 10–14 days and CSAs of developing
myeloid cells within the layer may also be visible. Non-adherent
cells can be assayed at feeding time (by counting with hemocy-
tometer of flow-cytometry for particular hematopoietic markers).
LTCs can typically be maintained for several weeks to months
before they decline.
Fig. 5 Morphology of MSC cultures. (a) A typical CFU-F that arises out of a single stromal precursor after 5–7
days of culture. (b) Confluent cultures that develop after 2 weeks or more of culture
3.5 Setting Up and Protocol for human MSC (hMSC) cultures is similar to that of LTCs,
Passaging of Human except that the medium used is DMEM replete with 10 % FCS.
MSC Cultures
1. Plate cells in densities as described above for LTCs.
2. Fibroblast-appearing MSCs should be evident 24–48 h after
plating although the preponderance of non-adherent hemato-
poietic cells might make initial visualization difficult.
3. About 48 h after plating, remove supernatant cells by suction-
ing (with sterile precautions) in the tissue culture hood. The
cultures may be rinsed with HBSS if the hematopoietic cells
form clumps. Refeed with equal amount of fresh medium.
4. CFU-Fs become rapidly evident in the next 3–5 days (Fig. 5a)
and cultures become confluent in about 2 weeks from when
they were plated (Fig. 5b).
5. Since primary cells have contact inhibition, MSCs could be
maintained for several weeks after reaching confluence with-
out splitting cultures; but to expand their numbers, the MSC
cultures can be split at a 1:3 ratio after they reach 75–80 %
confluence.
6. To split, rinse cultures 2–3 times with PBS–EDTA (half the
volume of tissue culture medium) after the medium is suc-
tioned off. Then add trypsin–EDTA (1 ml of 0.25 % or 4 ml of
0.05 % trypsin) and return to 37 °C.
7. After 5 min, determine how dislodgeable the cells are in
response to initial trypsin–EDTA treatment. If needed, the ini-
tial trypsin–EDTA can be suctioned off, an equal volume
added, and the plate returned to 37 °C (this will allow for the
initial trypsin to presumably loosen up the extracellular matrix
and the second trypsin to loosen the cell-to-cell adhesions).
8. After another 5 min, add 2 ml of FBS (or bovine calf serum
which is adequate to inactive trypsin but is much cheaper
than FBS) to inactivate the trypsin. Scrape the cells off the plate
with a sterile cell scraper and transfer cells to a 50 cc conical.
9. Wash plate with 5–10 ml of PBS–EDTA to collect cells that
may have been left behind.
10. Spin cells at 400 × g force for 5 min. Remove supernatant by
suctioning.
11. Resuspend pellet by “racking.” Add requisite amount of medium
(threefold if splitting 1–3) and plate to adherent plastic plates.
12. MSCs will maintain exponential growth typically for at least
4–5 passages and slow down after that. The characteristics of
MSCs will become more homogeneous after each passage and
closer to fibroblasts derived from any other tissue site after a
few passages.
3.6 Flow-Based Although a variety of immune-phenotypic markers have been used

ISOLATION OF MSC BY to define functional subtypes within marrow stromal cells, we have
CD146 high Expression found CD146 as initially described by Sacchetti et al. to be particu-
larly useful [17]. Commercially available CD146 antibodies are
robust reagents that can be used for immune-histochemistry
(Fig. 6a, b) and flow-cytometry (Fig. 6c). In our experience, sort-
ing cells based on CD146 positivity does correlate to expression of
other stem cell niche-associated genes such as CXCL12 and
Angiopoietin1 as described by Sacchetti et al. in their original
description of CD146+ self-renewing osteoprogenitors [17, 48].
In contrast to Sacchetti et al. (who performed flow-based sorting
on whole BMMNC), we performed flow-based sorting on hMSC
expanded for 1–2 passages [48]. This was done to increase the
proportion of stromal cells being sorted (allowing for most hema-
topoietic cells to be washed after the stromal precursors attach to
the plastic dishes) and consequently decrease the total of number
of cells needed to be sorted and reagents used. Although stromal
cells have likely changed their function and behavior even by this
short culture period, our group has reported that the CD146+
fraction of cells continue to express HSPC niche-associated genes
(CXCL12, Angiopoietin1) at a higher level when compared to
CD146-negative fraction [48]. CD146 has also been found to be
strongly expressed in the human stromal cell line Hs27a which is
functionally similar to the CD146+ osteoprogenitor fraction as
defined by Sacchetti et al.; Hs5 in comparison is CD146 negative
as would be consistent with its function [17, 48].
Flow-cytometry and flow-based sorting of adherent cells such
as fibroblasts and stromal cells are not straightforward and certain
important distinctions between hematopoietic cells need to be
kept in mind. Stromal cells, consistent with their fixed state in vivo
and adherent state in vitro, are not physiologically meant to flow
through narrow capillaries without blocking them and conse-
quently are difficult to easily sort into different populations while
Fig. 6 CD146 expression in primary stromal cells and its use in sorting stromal cells. (a and b) Immune histo-
chemistry (IHC) for CD146 expression of normal human bone marrow (Panel A is an isotype antibody control).
CD146-positive cells are present in a perivascular distribution, a location consistent with other models where
the HSPC niche might reside. (c) Flow-cytometry analysis of MSC cultures after 1 passage (10–14 days of
culture). A variable proportion of cells are CD146 positive and they inhabit continuum of antigen expression.
Two MSCs set up from separate donors are shown. (d) Typical flow sorting results from CD146-based sorting
of MSCs. Approximately 35 % of cells are deemed CD146 positive (presort, top histogram), and sorted to nega-
tive (middle histogram) and CD146 negative (bottom histogram) using a FACS-ARIA sorter (Beckton Dickinson
and Company) with a 100 μM nozzle
preserving viability. Since these cells are typically larger, the use of
a larger nozzle (100 or 130 μm) for the sorting machine would be
ideal and most modern flow-sorters are equipped to do this. While
preparing the cells, it is important to avoid using trypsin to dis-
lodge the cells since CD146 is known to digest the CD146 epitope
(see Note 11). Finally, viability of cells after sorting is typically
about 50 % at best (again likely reflecting the non-physiological
nature of single-cell capillary flow for these cells) and if these cells
were to be replated for culture, the loss of viability needs to be
considered.
1. Wash cultures initially with PBS–EDTA 3 times. Typically ten
to twenty 100 mm dishes are used for one sorting experiment.
2. Add enough citrate saline solution to cover the bottom culture
(for 100 mm plates, 5 ml of solution). Return plates to 37 °C
incubator for 5 min.
3. Scrape cells off the plate with a sterile cell scraper. Move the
cells to a 50 ml conical. Wash the plate with an additional 5 ml
of PBS–EDTA to collect any remaining cells and add to the
conical tube.
4. Spin cells down at 400 × g force for 5 min. Remove supernatant.
5. Gently resuspend cells by “racking” the pellet. Add 5 ml of
PBS–EDTA and mix thoroughly with the pipetman several
times.
6. Wash a second time with PBS–EDTA and resuspend cells as
described above.
7. Determine if cells are significantly clumped together (primary
MSC cultures are often clumped together) and you may opt to
repeat the PBS–EDTA resuspension step once more. If not,
filter through a sterile 100 μm filter and proceed to counting
and antibody labeling.
8. Determine viable cell number with a hemocytometer count.
9. Resuspend up to ten million cells in a clear 5 ml Falcon tube
(#2058) in sterile FACS buffer in a total volume not to exceed
1 ml (see Note 12).
10. Add 10 μl of anti-CD146 antibody per one million cells.
Incubate at 4 °C for 30 min.
11. Wash three times by adding 4 ml of FACS buffer, spinning at
800 × g force for 5 min, and resuspending the cell pellet after
each wash.
12. After final wash, filter cells once more through a 100 μm filter
and resuspend in a final cell concentration of no more than one
million cells per ml (to prevent cellular clumping).
13. Perform FACS-based sorting of cells on sorting machine, pref-
erably with a 100 or a 130 μm nozzle. Ensure that the cells are
agitated often to prevent clumping. Typical results for FACS-

based sorting are shown in Fig. 6d.
14. After sort, spin down cells at 800 × g for 30 min, count with
hemocytometer, and proceed with specific experiment.
3.7 Isolation of MSC An alternate method to enrich for MSCs is by depleting the bone
by Depletion of CD45 marrow of hematopoietic cells. Most mature cells of hematopoietic
and CD14 origin are CD45 (pan leucocyte antigen) positive; some monocytes
and macrophages may not be removed by CD45 depletion alone
and depleting CD14-positive cells in addition further enriches
for stromal precursors. As in the CD146 protocol above
(Subheading 3.6), enriching for adherent cells only by plating cells
in plastic adherent plates for 24 h can reduce the total reagents that
would need to be used if unselected fresh BMMNC is used.
Availability of magnetic conjugated antibodies for CD45 and
CD14 makes this protocol easy to perform with magnetic separa-
tors such as Auto-MACS (described earlier) which we have typi-
cally utilized for this purpose.
1. Isolate BMMNC as per Ficoll gradient separation protocol
above.
2. Plate BMMNC at a density of 50 × 106 cells per 100 mm plate
in MSC medium (DMEM with 10 % FCS).
3. Next day, wash off the non-adherent cells 3 times with HBSS.
4. Add 5 ml of 0.25 % trypsin–EDTA and incubate at 37 °C for
5 min to remove adherent cells. Visualize cells under microscope
to ensure adequate trypsinization. Dislodge with cell scraper if
needed.
5. Rinse flask with 5 ml medium to inactivate trypsin and collect
cells in 15 ml conical.
6. Spin cells down at 1,200 rpm for 5 min.
7. Resuspend cells in AutoMACS buffer (80 μl for up to 107
cells). Add 20 μl each of CD45 and CD14 microbeads.
8. Incubate on ice for 20 min.
9. Wash with 4 ml of AutoMACS buffer after incubation.
10. Perform AutoMACS with “Deplete” protocol, once only.
11. Spin down the negative fraction at 400 × g and resuspend in
MSC medium.
12. Plate in 100 mm dishes or T75 flasks with 0.1–0.5 × 106 cells
per flask; if the yield is low, plate at lower densities as well.
13. Isolated fibroblast colonies should be visible starting days 2–3.
To remove all the non-stromal components, change medium
every 2–3 days for the first 2 weeks as described for MSC
cultures.
3.8 Stromal Stromal cell lines HS5 and HS27are stable and well-characterized
Cell Lines lines that can be used to approximate various tissue microenviron-
ments. They are easy to culture and can be manipulated with rela-
tive ease (at least when compared to primary MSC).
1. Pre-warm medium (RPMI-1640 supplemented with 10 %
FBS) in a 37 °C water bath. Aliquot 45 ml of medium to a
50 cc conical tube.
2. Thaw cryopreserved cell vial (typically containing 5 × 106 cells)
in a 37 °C water bath. As with other cryopreserved cells, the
rule is to “freeze slowly and thaw quickly” to avoid cryogenic
damage to cells.
3. Remove vials to tissue culture hood once the frozen medium is
loosening from the side of the vial. Open vial, and add about
1 ml of pre-warmed medium to vial to complete the thaw.
4. Add the thawed cell–medium mixture to the 50 cc conical tube
containing warm medium. Mix well and spin down cells at
400 × g for 5 min.
5. Suction off supernatant and plate cells to one T75 flask (or
100 mm plate) with 10 ml medium. Transfer to incubator.
6. Determine viability of cells the next day—all viable cells should
be attached and exhibiting fibroblast-like pleomorphic mor-
phology. Cells can be split in about 48 h (1:3) if they are nearly
confluent.
7. Cells can be maintained by replacing medium without splitting
for up to a week after attaining full confluence.
8. To freeze stromal cell lines, use RPMI-1640 supplemented
with 30 % FBS. Resuspend about 5 × 106 cells per ml of this
medium and aliquot to cryovials. Add tissue culture-grade
DMSO to a final volume of approximately 10 % (100 ml to a
1 ml suspension). Move to cryo-freezing containers (such as
“Mr. Frosty” that allows for graded temperature drop) and
place in −80 °C freezer. Move frozen vials to liquid nitrogen in
24–48 h for long-term storage.
3.9 Reverse While stromal cells are not particularly difficult to grow and propa-
Transfection of gate, they can be challenging to transfect transiently with plasmids
Stromal Cell Line with or small RNAs. When working with siRNAs to inhibit specific tran-
siRNA/ miRNA scripts or miRNA, low-efficiency transfection (less than 25 %) is
almost guaranteed to result in a failed experiment, since the down-
stream tests of changes in RNA/protein levels or function are
likely not be reflected in those cells which were not transfected.
Commercially available lipid preparations such as lipofectamine
2000 from Invitrogen have been successfully adapted to the high-
efficiency transfection of several adherent cell types (such as HEK-
293Ts, HeLa). Typical protocols for liposomal transfections for
cells add the lipid–RNA mix to already adherent cells. Stromal cells
are typically difficult to transfect with this methodology presum-
ably due to extracellular matrix that is laid down immediately by
freshly plated cells that make lipid-based transfection difficult. One
way around this difficulty is by “reverse transfection.” In reverse
transfection cells are added to the medium that already has the
lipid–RNA mixes; higher efficiency likely results from the cells
being exposed to liposomal mixture before they adhere and secrete
matrix proteins [48]. We use siRNA or miRNA at 5–10 nmol final
concentration, but can be optimized based on the knockdown
achieved. The following are for a 12-well plate, but can be scaled
up or down as needed. All reagents need to be brought to room
temperature before beginning the protocol (see Note 13).
1. In a 5 ml clear sterile Falcon tube (#2058), mix 100 μl
OptiMEM with 2 μl lipofectamine 2000.
2. In a separate tube, mix 100 μl OptiMEM with 0.625 μl of
5 nmol miRNA/siRNA.
3. Let these mixtures stand for 5 min, and then gently mix
together. Keep for 10–15 min.
4. In the interim, plate 500 μl of medium without antibiotics
(usually RPMI with 10 % FCS) in a 12-well plate. Return to
TC incubator at 37 °C for 10–15 min.
5. Add 200 μl of the lipofectamine–RNA–OptiMEM mixture to
the 12-well plate slowly.
6. Return to incubator for another 10–15 min, total of about
30 min.
7. Suspend stromal cells (80,000–100,000) in 500 μl of antibiotic-
free medium (RPMI with 10 % FCS).
8. Add the cell suspension drop by drop to the well containing
the lipid mixture/medium. Gently tilt spread evenly (avoid cir-
cular swirling as this will allow for cells to congregate at the
center of each plate). Return to incubator.
Change medium (RPMI-1640 with 10 % FBS and antibiot-
ics) after 6 h or overnight incubation.
9. We typically can get transfect up to 80 % of the cells with the
above protocol (when using anti-GFP siRNA or plasmid for
controls, Fig. 7).
3.10 Concluding Mesenchymal stromal cells (MSC) can be isolated from bone mar-
Remarks and row with relative ease. Bone marrow screens which are typically
Summary discarded by-products of bone marrow harvests are an excellent
source of marrow cells and should be considered by investigators
with access to large clinical centers. The foremost consideration
while working with MSCs is to realize the wide variability of almost
all biological characteristics amongst MSCs established from vari-
ous donors. This is partly due to the complex nature of the culture
Fig. 7 Reverse transfection of stromal cell lines. (a) Stromal cell line HS27a stably expressing green fluorescent
protein (GFP) was transfected with control scrambled siRNA and visualized by inverted fluorescent microscopy
after 48 h. (b) Hs27a-GFP cell lines transfected with anti-GFP siRNA showing marked reduction in GFP expres-
sion of most cells
itself: stromal cells inhabit a continuum of differentiation from the

earliest stromal precursor capable of multilineage differentiation to
mature mesenchymal cells (osteoid, adipocytic chondroid, etc.).
Add to this the presence of macrophages and other hematopoietic
cells and the reason for wide variations becomes clear. Flow-based
sorting for CD146 has proven to be a useful technique in sorting
those earlier stromal precursors that both define CFU-Fs as well as
support hematopoietic stem and precursor cells. It is important to
recognize that stromal cells, consistent with their noncirculating
and non-transplantable biology in vivo, do not fare well with flow-
based sorting and hence special precautions have to be undertaken
while sorting them. The use of well-characterized stromal cell lines
can overcome the problems of primary MSC heterogeneity and
should be considered when mechanistic studies of the stromal
microenvironment are undertaken, although validation with pri-
mary MSC would be needed.
4 Notes
1. Aliquots of frozen marrow mononuclear cells are available for

purchase through the NIH-NIDDK-sponsored Core Centers
for Excellence in Hematology (CCEH) for investigators in the
USA at much reduced prices compared to commercial vendors
and this eliminates most of the regulatory concerns. Please visit
the CCEH Web site at http://cceh.fhcrc.org for further details.
2. Although immediate processing would be ideal, we have suc-
cessfully generated robust MSC cultures from screens pre-
served at 4 °C or at room temperature for up to 48 h (allowing
for transport between collaborator’s laboratories).
3. Both Hs27a and Hs5 are available from American Type Culture
Collection (ATCC, Manassas, VA) or can be directly obtained
from Beverly Torok-Storb laboratory in Seattle, WA.
4. We have used the FACS Aria sorter (Beckton, Dickinson and
Company, or BD) for our studies but multicolor sorters from
other manufacturers such as Beckman Coulter should work
just as well for these applications.
5. Several varieties of instrumentation are available for separation
of cells. The AutoMACS automated system is particularly use-
ful for larger cell numbers and can sort cells maintaining asep-
tic status and it is the system we use routinely. This instrument
is also typically available through flow-cytometry core facilities.
The protocol could be optimized for use with other magnetic
sorters with relative ease.
6. Since FBS is a complex biological product mass-produced as a
side-product from slaughterhouses, biological variability from
batch to batch is inevitable and it is recommended to screen lots
of FBS for performance in the particular biological assay of inter-
est. Commercial suppliers often provide small (~50 ml) aliquots
of FBS for testing to individual laboratories and hold those lots
for several weeks to allow for testing. Batches screened by the
commercial suppliers (deemed appropriate for MSC growth) are
also available directly albeit at typically much higher costs.
7. The horse serum needs to be specifically screened for the
ability to support LTCs and not MSCs.
8. Avoid delays in anticoagulant-free state given that marrow cell
pellets have powerful pro-coagulants activated and could result
in clots and clumps.
9. With complete or near-complete hemolysis, the supernatant
should be wine red in color and the cell pellet is pale or pinkish
but not frank red. If hemolysis is inadequate steps 1–3 can be
repeated 1–2 times (further hemolysis is unlikely to be helpful)
keeping in mind not to allow exposure to hemolytic buffer for
more than 5 min each time.
10. Recently, reports have indicated that Ficoll at 1.073 may be
more suitable for MSC progenitor isolation although this
observation has not been rigorously validated.
11. Protocols that do not use trypsin tend to leave primary MSCs
in a clumped state, so extra precaution needs to be taken to get
the cells into single-cell suspension.
12. Given the extreme stickiness of primary stromal cells, it would
be ideal to limit total cells in the approximately five million cells.
13. Also note that transfection needs to be performed in antibiotic-
free medium as the presence of antibiotics will reduce transfec-
tion efficiency and cell viability.
Acknowledgments
This work was supported in part by NIH grants DK073701,

DK082757, HL104070, DK082783, HL099993, and DK056465,
Bethesda, MD, USA.
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Mesenchymal stem cells: revisiting history, Blood 85:997–1005
Chapter 9
Detection In Vitro and Quantitative Estimation of Artificial

Microterritories Which Promote Osteogenic Differentiation
and Maturation of Stromal Stem Cells
Igor A. Khlusov, Natalya M. Shevtsova, and Marina Yu. Khlusova
Abstract
Extracellular matrix can regulate multipotent mesenchymal stromal cells (MMSC) differentiation, with
potential applications for tissue engineering. A relief of mineralized bone takes essential effect on MMSC
fate. Nevertheless, delicate structure and a hierarchy of niches for stromal stem cells and its quantitative
parameters are not practically known. Here, we describe the protocol to define the basic approach provid-
ing a guiding for in vitro identification of quantitative features of artificial calcium phosphate niches which
promotes osteogenic differentiation and maturation of stromal stem cell.
Key words Prenatal stromal cells of human lung, Calcium phosphate surface, Short-term culture,
Artificial osteogenic niches, Alkaline phosphatase, Acid phosphatase, Osteocalcin,
Immunocytochemistry, Light microscopy, Scanning electron microscopy
1 Introduction
To explain a fundamental and contradictory phenomenon of self-

maintenance and differentiation processes of hematopoietic stem
cells (HSC) R. Schofield put forward a hypothesis on a hematopoi-
etic niche (specialized stem cell microenvironment) as an essential
matter for the maintenance of stem cell phenotype [1]. The com-
mon features, structure, and functions of the stem cell niche and
highlight important molecular niche signals are summarized from
Drosophila to mammals. Both environmental regulatory signals
and intrinsic programs are required to maintain stem cell proper-
ties and to direct stem cell proliferation and differentiation [2].
Our point of view allows selecting modern achievements in
this field. First of all, a hierarchy of niches for HSC self-maintenance,
proliferation or differentiation [3] and their some topographical
localization are proposed. Nevertheless, delicate extracellular
structure of niche and its quantitative parameters are not
103
104 Igor A. Khlusov et al.
practically known. The trabecular bone surface and osteoblastic

cells are the key components of bone marrow HSC niche [4]. So,
osteoblastic (endosteal) niches promote quiescent HSC state and
perivascular ones do an active hematopoietic cells proliferation [5].
Multipotent mesenchymal stromal cells (MMSC) are treated as
important cell component of HSC niche [6]. At the same time, a
discussion about an existence of MMSC niches themselves just
appears. A.S. Curtis and M. Varde [7] supposed the most impor-
tant role of surface topography and geometry in a determination of
cell behavior as early as in 1964. There is evidence that extracellular
matrix (ECM) alone can regulate MMSC differentiation, with
potential applications for tissue engineering. For instance, a relief
of mineralized bone takes essential effect on cell fate [3]. Bone
ECM synthesized by osteoblasts in vitro on titanium scaffolds can
increase MMSC osteogenic markers, such as alkaline phosphatase
activity and calcium deposition [8].
Designing artificial matrices that can mimic the tissue microen-
vironment and regulate the appropriate differentiation and matu-
ration of stem cells is a promising approach to therapeutic
applications [9]. According to [10], scientists are only at the begin-
ning of understanding of ECM effect. For all this, attempts of bio-
mimetic modeling of stem cell niches by means of artificial materials
have been already made [11]. A behavior of cells in such construc-
tions is studied (see ref. [12]). Nevertheless, no specific matrix
components have been identified that help to maintain MMSC
state, as a niche matrix would to [9].
In this connection, an effect of relief features of model ECM on
structural–functional state of MMSC and on remodeling bone/
bone marrow system is of great interest. We obtained in vivo repro-
ducible results of ectopic osteogenesis test on relief CP surfaces
mimicking the bone mineral matrix [13]. Pilot experimental data
about a possibility to find the microterritories (“artificial niches”)
for MMSC osteogenic differentiation on relief CP surfaces were
recently obtained in vitro [14]. We hope this protocol defines the
basic approach providing a guiding for further identification of
quantitative features of stromal and hematopoietic stem cell niches.
2 Materials
Prepare and store all reagents and samples at room temperature

(unless indicated otherwise). Prepare all solutions using ultrapure
water (prepared by purifying deionized water to attain a sensitivity
of 18 M Ω cm at 25 °C) and analytical grade reagents.
2.1 Samples 1. 10 mm × 10 mm × 1 mm composite plates from technical pure

titanium with bilateral rough (Ra = 1–6 μm) calcium phosphate
(CP) coating (see Note 1).
2. Prenatal stromal cells of human lung (HLPSC) (see Note 2).
Artificial Osteogenic Microterritories for Stromal Stem Cells 105
2.2 Cultural 1. Cultural osteogenic medium: 80 mL DMEM/F12 (1:1)

Components (Gibco, USA), 20 mL fetal bovine serum (Gibco, USA),
50 mg/L gentamicin (Invitrogen, UK) and freshly added ster-
ile solutions in final concentrations of 280 mg/L L-glutamine
(Sigma, USA), 10 mM β-glycerophosphate (Gibco, USA),
10 μM dexamethasone (KPKA, Slovenia), 50 mg/L ascorbic
acid (Sigma, USA) (see Note 3). Store at 4 °C.
2. Trypan blue solution: dissolve 40 mg trypan blue in 10 mL
Hank’s balanced salt solution. Store for 1 month.
3. 24-Well plastic plates (Orange Scientific, Belgium).
4. Plastic screw cap tubes (SCT; 15 mL): conical bottom, pre-
sterilized (Axygen Scientific, Union City, CA, USA).
5. Supernatants of 4th day cell culture. Store the aliquots at −20 °C.
2.3 Fixative 1. Formalin: 37 % paraformaldehyde solution in water.

Components 2. Formalin chamber.
3. Glutaric dialdehyde (C5H8O2): 2.5 % solution in phosphate
buffer solution (PBS) (see Note 4). Store a few days at 4 °C.
2.4 Components for 1. Naphthol AS-BI phosphate (NASBIP, C18H15NO6P, m.w.

Alkaline Phosphatase 452.21) (Lachema, Czech Republic).
(ALP) Staining 2. Fast blue PP salt (C15H15N3O3·BF4, m.w. 372.10) (Lachema,
Czech Republic).
3. N,N-dimethylformamide (DMF, C3H7ON, analytical) (Sigma-
Aldrich, USA).
4. 0.1 N Hydrochloric acid (HCl).
5. Buffer: 0.2 M Tris. Add 100 mL water to a 1-L graduated glass
cylinder (see Note 5). Weigh 24.3 g Tris(hydroxymethyl)ami-
nomethane and transfer to the cylinder. Add water to a volume
of 1 L. Store at 4 °C.
6. Tris-hydrochloric acid buffer: 0.2 M Tris–HCl, pH 8.7. Mix
25 mL Tris with 12 mL 0.1 N HCl. Add water to a volume of
100 mL. Adjust pH to 8.7. Store at 4 °C.
7. Substrate working solution (SWS): Mix to dissolve 10 mg
NASBIP in 0.5 mL of DMF (see Note 6). Add 50 mL of Tris–
HCl buffer and 50 mg of fast blue PP salt. Shake thoroughly
before dye complete dissolution. Filter the mixture through
Corning filter (see Note 7). Prepare ex tempore.
8. 24-Well plastic plate.
2.5 Components for 1. Naphthol AS-BI phosphate (NASBIP, C18H15NO6P, m.w.

Acid Phosphatase 452.21) (Lachema, Czech Republic).
(ACP) Staining 2. N,N-dimethylformamide (DMF, C3H7ON, analytical)
(Sigma-Aldrich, USA).
3. 0.1 N Hydrochloric acid (HCl).

4. Sodium acetate solution (SAS; 0.1 N): Weigh 4.1 g SAS
(CH3COONa) and dissolve in 0.5 L water.
5. Sodium nitrite solution (SNS; 4 %): Dissolve 1 g SNS (NaNO2)
in 25 mL water.
6. Pararosaniline solution (PRN; 4 %): Dissolve 1 g basic fuchsin
in 25 mL of 0.1 N HCl.
7. Prepare solution A: Mix to dissolve 25 mg NASBIP in 0.5 mL
of DMF (see Note 6) and add 40 mL SAS. Prepare ex
tempore.
8. Prepare solution B: Mix 0.4 mL of PRN with 0.4 mL of SNS
(see Note 8). Prepare ex tempore.
9. Substrate working solution (SWS): Mix solution A and solu-
tion B (see Note 9). Prepare ex tempore.
10. Filter the mixture through Corning filter (see Note 7). Prepare
ex tempore.
2.6 Immunocyto- 1. Primary antibodies (Epitomics Inc., USA): rabbit polyclonal

chemical Components anti-human IgG (1:100) diluted in PBS with 0.09 % sodium
for Osteocalcin (OC) azide. Store the aliquots at −20 °C.
Detection 2. Histofine Simple Stain MAX PO MULTY (Nichirei Biosciences
Inc., Japan): universal immunoperoxidase anti-rabbit and anti-
mouse polymer. Store at 4 °C.
3. PBS (negative control): Dissolve 7.75 g NaCl, 1.5 g K2HPO4,
0.2 g KH2PO4 in 1 L water, pH 7.6. Store at 4 °C (see Note 4).
4. H2O2 solution: 3 % H2O2 in water (see Note 4).
5. Histofine DAB-3S kit peroxidase chromogen/substrate kit
(Nichirei Biosciences Inc., Japan): Mix 1 drop of reagents A
with 1 mL water (see Note 10). Add 1 drop of reagents B to
reagents A solution. Add 1 drop of reagents C to reagents A
and B mixture and mix. Store for 2 weeks in dark place at 4 °C
(see Note 11).
6. Blocking solution: Normal horse serum from Novostain uni-
versal detection kit (Novocastra Laboratories Ltd., UK).
2.7 Light Microscopy 1. Olympus GX-71 inverted metallographic microscope.
2.8 Scanning 1. Osmium tetroxide solution (OsO4; OTS; 2 %): Dissolve 2 mL

Electron Microscopy OsO4 in 98 mL water (see Note 12). Store in dark place at
Components 4 °C, in a bottle wrapped with aluminum foil and with ground
stopper.
2. Osmium tetroxide solution (OTS; 1 %): Dissolve 2 % OTS in
equal volume of PBS. Use ex tempore.
3. Acetone (С3H6O).
4. Alcohol (ethyl hydroxide, С2H5OH; 30°): Mix 31 mL of 96°
ethanol with 69 mL water.
8. Alcohol (ethyl hydroxide, С2H5OH; 100°): Add 96° ethanol
2–3 times to heat-treated copper sulfate (CuSO4) crystals (see
Note 13).
10. Fume hood.
11. Phillips S 515 or Zeiss EVO 50 XVP scanning electron micro-
scopes (SEM).
3 Methods
Carry out all procedures at room temperature unless otherwise

specified.
3.1 Short-Term 1. Prior to use, sterilize the samples of CP-coated titanium plates
Cultivation with by dry-heart manner (Binder FD 53, Germany) for 60 min at
Composite Samples 160 °C.
2. Place one CP-coated titanium specimen in each plastic well
(1.86-cm2 area) of 24-well plate (Orange Scientific, Belgium).
Use plastic wells without composite samples as control of cell
growth (see Note 14).
3. Prepare HLPSC suspension in osteogenic medium with a con-
centration of 3 × 104 viable karyocytes/mL. Estimate cell via-
bility by means of 0.4 % trypan blue staining (see Note 15).
4. Add cell suspension in a volume of 1 ml per well of plastic
plates with or without CP-coated titanium specimens. Use
plastic wells with cultural medium probes along to estimate
initial levels of molecular metabolites (see Note 14).
5. Incubate the cell culture for 4 days in a humidified atmosphere
of 95 % air and 5 % CO2 at 37 °C (see Note 16).
6. Collect the suspensions of nonadherent cells from wells into
SCT.
7. Centrifugate SCT at 500 × g for 15 min.
8. Collect the supernatants from SCT and freeze immediately
0.9 mL aliquots at −20 °C before immunoenzyme and bio-
chemical tests.
9. Determine osteocalcin concentration in aliquots according to

Osteometer BioTech A/SN-MID Osteocalcin One Step
ELISA (Nordic Bioscience diagnostics, Denmark) protocol
(see Note 17).
10. Determine calcium and inorganic phosphate concentrations
and ALP activity in aliquots by means of standard colorimetric
method [15] with the help of Konelab™/T Series protocols.
They can be purchased from Thermo Fisher Scientific Inc.,
USA (see Note 17).
3.2 Cells Fixation 1. Remove composite samples from plastic wells. Dry the samples
and plastic plates with adherent cells in the air for 24 h.
2. Fix composite samples and plastic plates with adherent cells in
formalin vapors for 30 s to use the (immuno)cytochemical
staining probes (see Note 18).
3. Fix other composite samples with adherent cells in glutaric
dialdehyde solution for at least 24 h at 4 °C to use SEM.
4. Wash with water for 1 min and air-dry.
5. Store fixed samples at 4 °C.
3.3 Cytochemical 1. Place composite samples with adherent cells to pure plastic
Staining for ALP by wells of a 24-well plate (see Note 14).
Diazocoupling 2. Use plastic wells with adherent cells after removing composite
Technique samples as control of ALP staining.
3. Add 0.5 mL of SWS into each well of a 24-well plate.
4. Incubate in dark at 37 °C for 45 min.
5. Aspirate SWS and rinse 3 × 1 min in distilled water.
6. Air-dry composite samples and 24-well plate.
7. ALP staining criteria (Fig. 1): blue sites of cytoplasmic enzy-
matic activity (see Note 19). Positive staining control: neutro-
cytes of human blood.
3.4 Cytochemical 1. Place composite samples with adherent cells to pure plastic
Staining for ACP by wells of a 24-well plate (see Note 14).
Diazocoupling 2. Use plastic wells with adherent cells after removing composite
Technique samples as control of ALP staining.
3. Add 0.5 mL of SWS for each well of a 24-well plate.
4. Incubate in dark at 37 °C for 2 h.
5. Make the procedures as in previous Subheading 3.3.
6. ACP staining criteria (Fig. 2): pink sites of cytoplasmic enzy-
matic activity (see Note 20). Positive staining control: neutro-
cytes of human blood.
Fig. 1 Fourth day culture of prenatal stromal cells of human lung (HLPSC) on
plastic surface of 24-well plate. Cells with alkaline phosphatase (ALP) under-
staining are marked by black arrows. Magnification, 350×
Fig. 2 Acid phosphatase (ACP) stained HLPSC on calcium phosphate surface of

composite samples. ACP-positive cells are marked by black arrows. Magnification,
1,000×
3.5 Immunocyto- 1. Place composite samples in pure 24-well plastic plates

chemical Detection (see Note 21).
of Osteocalcin 2. Add 0.2 mL of H2O2 solution for 5 min to deactivate an
endogenous peroxidase.
3. Wash with PBS for 5 min.
4. Blot excess liquid from samples.
5. Add 0.2 mL of blocking solution for 10 min (see Note 22).
Fig. 3 Osteocalcin (OC) stained HLPSC on calcium phosphate surface of compos-

ite samples. OC-positive cell is marked by black arrow. Magnification, 1,000×
6. Blot excess solution from samples.

7. Incubate the samples with 0.2 mL of primary antibodies or
negative control in a humidified atmosphere for 45 min at
37 °C (see Note 23).
8. Wash the samples 3 × 1 min with PBS.
9. Blot excess buffer from samples.
10. Incubate the samples with 0.2 mL of Histofine Simple Stain
MAX PO MULTY for 30 min.
11. Wash the samples 3 × 5 min with PBS.
12. Blot excess buffer from samples.
13. Incubate the samples with 0.2 mL of Histofine DAB-3S kit
peroxidase chromogen/substrate kit with microscopic control
of staining (see Note 24).
14. Wash the samples 3 × 5 min with PBS and air-dry.
15. Store staining samples at 4 °C in dark place.
16. OC staining criteria (Fig. 3): brown sites of stained cells.
3.6 Reflecting Light 1. Use Olympus GX-71 inverted metallographic microscope to

Microscopy estimate ALP, ACP, or OC stained cells localization at a mag-
nification of 500× and 1,000×.
2. Result: ACP-positive cells (see Fig. 2) are located on spherolites
forming relief of composite samples with rough CP coating.
ALP stained cells (Fig. 4) populated sockets of CP surface
(see Note 25).
Fig. 4 ALP stained HLPSC on calcium phosphate surface of composite samples. (a) Blue cell sites stained with
fast blue PP salt. (b) Cell site with garnet color stained with fast garnet Gbc salt. ALP-positive cells are marked
by black arrows. Magnification, 500×
3.7 Scanning 1. Place composite samples with adherent cells in pure 24-well
Electron Microscopy plastic plates (see Note 26).
2. Add 1 mL of 1 % OTS for 30 min.
3. Wash the samples 2 × 5 min with 1 mL of PBS.
4. Dehydrate the samples through a graded series of alcohols
(30°; 50°; 70°; 90°; 100°) for 15 min with each alcohol
concentration.
5. Rinse the samples 2 × 15 min with acetone (see Note 27) and
air-dry.
6. Store the samples at 4 °C in dark place before SEM.
7. Examine the samples in Phillips S 515 or Zeiss EVO 50 XVP
SEM at an accelerating voltage of 15 kV.
8. Result: (1) spreaded cells with rounded shape (Fig. 5) or fibro-
blast-like cells on the surface of samples (Fig. 6); (2) three-
dimensional (3D) shape of osteoblast-like cells in the sockets
(Fig. 7) (see Note 28).
4 Notes
1. CP coatings are applied on titanium substrate by means of

anode-spark (microarc) oxidation method in phosphoric acid
solution containing suspension of synthetic hydroxylapatite
nanoparticles with stoichiometric composition Са10(РО4)6(ОН)2
(see ref. [16]). Composite samples size must be no more than
10 mm × 10 mm × 1 mm to be placed in wells of 24-well plastic
Fig. 5 Scanning electron microscopy (SEM) image of rounded HLPSC on calcium phosphate surface of com-
posite samples. Cell is marked by an arrow. Magnification, 5,000×
Fig. 6 SEM image of fibroblast-like HLPSC on calcium phosphate surface of composite samples. Cell is marked
by black arrows. Magnification, 5,000×
Fig. 7 SEM image of osteoblast-like HLPSC in the socket of calcium phosphate

surface. Cells are marked by black arrows. Magnification, 1,250×
plates. Roughness (Ra index, μm) of CP surfaces is evaluated by

means of Talysurf 5-120 measuring system (Taylor-Hobson,
UK, resolution 10 nm). No composite samples with Ra > 6 μm
use because of limited optical visualization of their surface relief.
2. HLPSC strain FL-42 was primarily isolated from lung of
11-week human fetus in 2003 (Stem Cell Bank Ltd., Tomsk,
Russia). Aliquots are CD34-CD44+ adherent cells with differ-
ent shape and size (see Fig. 1), maintaining diploid karyotype
and oncological safety during ex vivo passages. Cells are free
from viral (HIV, hepatitis, herpes, etc.), bacterial (syphilis,
mycoplasma, chlamydia, etc.), and fungous agents. After being
unfrozen, 91–93 % cells viability of HLPSC is determined
according to ISO 10993-5 test with 0.4 % trypan blue.
Stromal stem cells (SSC) represent heterogeneous multi-
cellular pool that occurs in embryonic tissues [17] and adult
human lung [18]. In this connection, HLPSC culture can be
a source of regional SSC pool. HLPSC show fibroblast-like
morphology and sharp ALP activity in osteogenous medium
in 8th day culture on plastics (Fig. 8). HLPSC strain fails to
have existing (pre)osteogenic cells unlike bone marrow. No
HLPSC differentiate into osteoblast-like cells in short-term
(4th day) culture, but they are capable to do it in the presence
of CP materials (see ref. [14]). It allows considering the
HLPSC strain as a specimen to study SSC osteogenic differen-
tiation and maturation induced by artificial matrix.
Fig. 8 A confluent growth of fibroblast-like cells in 8th day HLPSC cultivation on

plastic surface. ALP staining with fast blue PP salt. Magnification, 120×
3. Fully supplemented osteogenous medium includes the

inductors of differentiation (as a rule, dexamethasone,
β-glycerophosphate, and ascorbic acid) which concentrations
are rather different in different authors (see refs. [17, 19]). We
use [20] protocol in our modification.
4. It is best to prepare glutaric dialdehyde solution, H2O2 one
and PBS fresh each time.
5. Having water at the bottom of the cylinder helps to dissolve
Tris relatively easily.
6. It is important to dissolve NASBIP crystals completely.
7. It is necessary to prepare yellow clear solution.
8. Solution B is slightly fading and sulfur dioxide is producing
following reagents mixing.
9. SWS will be hazy and will have crimson.
10. It is recommended in Novostain universal detection kit
instruction that the peroxidase substrate solutions be prepared
with glass distilled water. Deionized water may contain inhibi-
tors of peroxidase which can reduce sensitivity.
11. It is best to use a supernatant of final mixture.
12. All work should be done in a fume hood.
13. Spread heat-treated copper sulfate crystals to the bottom of
glass bottle with ground stopper. Fully dehydrated ethanol
does not change gray color of heat-treated copper sulfate crys-
tals. Use glass bottle with ground stopper to store 100° etha-
nol for a long time.
14. Use no less than 3 composite samples and control wells

(without composite samples) for each examination (ALP,
ACP, OC, SEM) of cell culture. Use no less than 3 probes
with cultural medium along (without cells) as well.
15. Mix cell suspension 1:1 with trypan blue solution. Count cells
on hemocytometer or using the Goryaev chamber. Viable cells
exclude trypan blue, while dead cells are permeable, take up
the dye and stain blue. Unfrozen HLPSC viability is no less
than 90 % usually. Avoid the exposure of cells to trypan blue
for a period longer than 20 min. In this case an increase in the
dead (blue stained) cell population occurs due to the dye
toxicity.
16. HLPSC achieve fibroblast-like morphology with ALP-positive
(blue stained) forms and a confluence in 8th day growth on
plastic surface (see Fig. 8). So, 4th day cell cultivation is
preferable.
17. ALP and OC are considered as general markers of osteoblasts
[21]. Extracellular calcium is estimated as a contribution of
bone mineral matrix into hematopoietic stem cell niche func-
tioning [22]. Relief CP coatings promoted ALP and OC
secretion, Ca and phosphate metabolism in HLPSC culture as
described [14]. In this connection, it is best to evaluate func-
tional status of HLPSC culture contacting with artificial sur-
face. According to cell culture indices, HLPSC interacting
with CP samples directly obtained advantage in display of their
osteoblast-like functional activity in comparison with cells on
plastic wells. ALP and OC secretion is directly increased with
a growth of CP surface roughness index [14].
18. Fixing cells longer than 1 min will result in the inactivation of
enzymes.
19. Do not use nuclei counterstain with hematoxylin or azure. It
will be very difficult to distinguish between ALP-positive and
negative cells on a surface of composite samples. ALP detec-
tion by diazocoupling technique with Naphtol AS-MX phos-
phate and fast garnet Gbc salt (C14H13N4·BF4, m.w. 324.11)
(Lachema, Czech Republic) may be used. Garnet color sites of
cytoplasmic enzyme activity will be revealed (see Fig. 4b).
20. Do not use nuclei counterstain with hematoxylin or azure. It
will be very difficult to distinguish between ACP-positive and
negative cells on a surface of composite samples.
21. It is best to test fresh fixed samples with adherent cells.
22. Blocking solution prevents false-positive stain because of non-
specific protein bounding. No differences were noted with or
without blocking solution when Histofine Simple Stain MAX
PO MULTY was used.
350
300
Square(S) of ALP staining, µm2 r = 0,939, p = 0,00001
250
200
150
100
50
-50
-100 0 100 200 300 400 500 600 700 800
S of sockets, µm2
Fig. 9 A regression curve of the squares of ALP-stained sites in HLPSC cells and surrounding sockets of rough
calcium phosphate surface
23. The length of incubation times depending on the concentra-

tion of primary antibody. Generally, optimal staining is
achieved with incubation times of 15 min to 1 h.
24. Development times may differ depending upon the level of
antigen, the intensity of the stain or the substrate used.
DAB-3S kit generally should be developed for 1–10 min. With
the help of light microscopy it is necessary to determine a
staining level (time interval) needed.
25. Composite samples with CP rough surfaces stimulated forma-
tion of three-dimensional culture of HLPSC. ACP-stained
cells are located on spherolites forming relief of CP coatings.
ALP-stained (see Fig. 4) and OC-stained (see Fig. 3) cells
(osteoblasts marker) are manifested in the sockets of CP sur-
face. Thus, osteoblast-like cells differentiate from HLPSC in
the sockets of CP relief. Such microterritories were named us
as artificial osteogenic “niches” (see ref. [14]). Final sizes of
sockets surrounding ALP-stained cells allow calculating the
squares (μm2) of ALP-stained cells and their artificial niches
with the help of ImageJ program. Close regression of squares
was estimated (Fig. 9).
In this connection, SALP/Sniche (%) index has been
described (see ref. [14]). It correlates directly with CP rough-
ness index (r = 0.91; p < 0.01). This protocol allowed us to
develop “niche-relief” conception for HLPSC osteogenic dif-
ferentiation and maturation. Rough CP surfaces have own
Fig. 10 SEM image of rough calcium phosphate surface. Spherolites with pores
are presented. Magnification, 1,250×
Fig. 11 SEM image of rough calcium phosphate surface. Interconnected sockets

are situated as dark fields. Magnification, 156×
structural sites (microterritories) which are named by “niche-

relief” and are able to accelerate in vitro HLPSC morphofunc-
tional changes into secreting osteoblasts [23]. Apparently,
artificial “niches” for promotion of HLPSC osteogenic differ-
entiation are a structural-functional concept.
26. Use a fume hood for each step of samples preparation.
27. Remove the samples from plastic wells. Rinse the samples by
acetone in glass.
28. SEM shows the irregularities of CP coating relief are represented
by spherolites (diameter up to 20–30 μm) and single or numer-
ous pores with diameter of 5–20 μm (Fig. 10). Interconnected
sockets are situated between spherolites (Fig. 11). Morphology
of rough CP coating simulates a structure of regenerating bone.

SEM confirms a formation of HLPSC 3D-culture and estab-
lishes the differences in cell morphological shape in diverse sites
of CP surface (see Fig. 5, 6 and 7).
Acknowledgments
The authors are deeply indebted to: professor Yu.P. Sharkeev and
E.V. Legostaeva Ph.D. (Institute of Strength Physics and Materials
Science, SB of RAS, Tomsk, Russia) for designing and digital imag-
ing of titanium specimens with calcium phosphate coating; K.V.
Zaitsev Ph.D. (Stem Cells Bank Ltd., Tomsk, Russia) for cell cul-
ture provision; Joint Use Center for Materials Science (Tomsk
State University, Tomsk, Russia) for microscopic equipment use.
This work was supported by the Federal Goal Program of
Russian Federation (grant No 8036).
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Chapter 10
The Prospective Isolation of Viable, High Ploidy

Megakaryocytes from Adult Murine Bone Marrow
by Fluorescence Activated Cell Sorting
Shen Y. Heazlewood, Brenda Williams, Melonie J. Storan,
and Susan K. Nilsson
Abstract
Mature megakaryocytes (MM) can be up to 65 μM in diameter and due to their size, viable and pure MM
populations have been difficult to isolate in large numbers. Here in, we report a fluorescence activated cell
sorting (FACS) method by which viable and pure populations of 8 N, 16 N, 32 N, and 64 N MM can be
isolated from murine bone marrow (BM).
Key words Megakaryocytes, Ploidy, Bone marrow, Microenvironment, Niche
1 Introduction
In adults, hemopoietic stem cells (HSC) reside in the BM where

extrinsic cues from hemopoietic and non-hemopoietic cells influ-
ence their fate decisions such as maintenance or differentiation into
mature blood cells [1]. MM, which originate from HSC, are pri-
marily known for the production of platelets, which are essential
for normal blood clotting and wound healing. MM are randomly
distributed in the BM (see Fig. 1) [2] and during megakaryocyte
maturation, DNA replication occurs without cytokinesis, resulting
in megakaryocytes of increasing ploidy up to 65 μM in size [3].
Previously published methods for the isolation of MM include
MM sedimentation through a bovine serum albumen (BSA) gradi-
ent [4, 5], direct aspiration from BM cell suspension [6], FACS
[7, 8], separation via a continuous Percoll gradient [9], MM
enrichment via immunomagnetic beads [10], and Botrocetin
agglutination [11]. However, these methods often required fixa-
tion of cells before processing, result in enriched but not pure MM
populations, or insufficient numbers of MM were isolated for fur-
ther experimentation [12–14]. We report using an anti-CD41
121
122 Shen Y. Heazlewood et al.
Fig. 1 Immunofluorescence image of an adult longitudinal femoral murine section.

Megakaryocytes are labeled with a FITC conjugated anti-c-Mpl antibody (green)
and the section counterstained with the nuclear dye DAPI (blue) (20×)
antibody and the DNA binding dye Hoechst 33342, viable, pure
8 N, 16 N, 32 N, and 64 N MM can be sorted from murine BM.
Lineage depleted central BM cells were sorted at ~5,000 events per
second using a 100 μM nozzle set at 20 psi. MM function was
unaffected, MM isolated in this manner are able to form pro-
platelet extensions whilst in culture.
2 Materials
2.1 Isolation 1. Adult C57Bl/6J (Ly5.2), GFP, or RFP mice, 6–8 week old
of Bone Marrow (see Note 1).
2. Sterile #11 surgical blade and #3 handle.
3. Phosphate buffered saline (PBS): pH 7.2, 310 mOsm
(see Note 2) supplemented with 2 % Se (Serum): Defined
bovine calf serum, iron supplemented.
4. 1 ml syringes attached to 21-gauge and 23-gauge needles to
flush central marrow from bones.
5. 50 ml conical tubes for collection of BM.
6. 100 μm nylon cell strainers.
7. Hemocytometer and microscope equipped with phase contrast
or an automated cell counter. We use the Sysmex KX-21N
(Japan).
2.2 Immunomagne- 1. Lineage depletion antibody cocktail: a mixture of purified rat

tic Cell Separation anti-mouse antibodies recognizing the cell surface antigens:
B220, GR-1, and MAC-1 (BD Pharmingen, USA) (see Note 3).
Isolating Viable High Ploidy Megakaryocytes 123
2. Antibody dilution buffer: PBS supplemented with 2 % Se.

3. PBS supplemented with 2 mM EDTA and 0.1 % (w/v)
fraction V BSA, pH 7.4.
4. Dynabeads for magnetic labeling of the cells: Sheep anti-rat
IgG beads, 4.5 μm diameter, 4 × 108 beads/ml (Dynal Biotech
ASA, Oslo, Norway).
5. Magnets: We use Dynal MPC-S for 20 μl to 2 ml samples and
MPC-L for 5–14 ml samples.
6. Tube rotator or similar suspension mixer: allowing both tilting
and rotation at 4 °C for Dynabead incubation step (We use a
MACSmix Tube Rotator placed in a fridge).
7. Falcon polypropylene round bottom tubes: 1.7, 5, and 14 ml
tubes.
2.3 Fluorescence 1. Fluorescent conjugated antibodies: purified fluorescein iso-

Activated Cell Sorting thiocyanate (FITC) or phycoerythrin (PE) conjugated to rat
anti-mouse CD41 (Integrin α11b chain, BD Pharmingen, USA)
(see Note 1).
2. Antibody dilution buffer: PBS-2 % Se.
3. Hoechst 33342 (Molecular Probes, Eugene, USA).
4. Falcon polypropylene 5 and 50 ml conical tubes.
5. Water bath maintained at 37 °C.
6. Flow cytometer with sorting capability. We use a Cytopeia
Influx 516SH cell sorter (Cytopeia, WA, USA) equipped with
5 solid state lasers (355, 405, 488, 561, and 633 nm). Band
pass filter settings for the detection of fluorescence for FITC/
GFP, PE/RFP, and Hoechst are 528 ± 19, 605 ± 20, and
460 ± 25, respectively. We use a 100 μm nozzle and sort at
20 psi (see Note 4).
7. Sheath fluid: Isoton II (Beckman Coulter).
8. Samples are collected in 1.7 ml microtubes.
9. Hemocytometer.
10. Microscope equipped with phase contrast and fluorescence.
3 Methods
3.1 Isolation 1. Sacrifice three mice by cervical dislocation. Dissect and clean
of Bone Marrow femurs, tibias, and iliac crests (see Note 5).
2. Using a 1 ml syringe containing PBS-2 % Se attached to a
21-gauge needle, repeatedly flush out the central marrow into
a 50 ml centrifuge tube containing 40 ml of PBS-2 % Se by
inserting the needle in turn into each epiphysis of the femoral
shaft and the knee epiphysis of the tibia. To flush the iliac crest,
insert a 1 ml syringe fitted with a 23 gauge needle and flush the
BM into the same 50 ml centrifuge tube. Wash the cells by
centrifuging at 300 × g for 5 min at 4 °C (see Note 6).
3. Decant supernatant and resuspend the cell pellet in 10 ml
PBS-2 % Se.
4. Filter the cell suspension through a 100 μm nylon cell strainer
into a fresh 50 ml conical tube (see Note 7).
5. Dilute cells to 40 ml with PBS-2 % Se and perform a cell count.
3.2 Immuno- 1. Pellet cells by centrifugation at 300 × g for 5 min, 4 °C.

magnetic 2. Decant supernatant.
Cell Separation
3. Resuspend cell pellet at 1 × 107 cells/100 μl in an optimally
3.2.1 Immunolabeling pre-titered cocktail of lineage antibodies (see Note 8). We use
Cells with a Cocktail 2 μg/ml of anti-B220 and 1 μg/ml of anti-GR-1 and
of Lineage Antibodies anti-MAC-1.
4. Incubate cells for 15 min on ice.
5. Wash labeled cells in PBS-2 % Se by centrifuging at 300 × g for
5 min, 4 °C to remove unbound antibody.
6. Remove supernatant and resuspend the cell pellet in PBS
supplemented with 2 mM EDTA and 0.1 % BSA at
1 × 108 cells/ml.
3.2.2 Dynabeads 1. Vortex Dynabeads.

Washing Procedure 2. The optimal Dynabead to cell ratio used in this protocol has
been established as half a bead per cell, with the depletion
repeated with the same number of beads (see Note 9). Dispense
beads for both steps into a 1.7 ml or 5 ml tube and follow the
washing procedure as outlined below (see Note 10):
(a) Add 1 ml of PBS 2 mM EDTA and 0.1 % BSA to the tube
and mix.
(b) Place the tube on the magnet for 1 min before removing
and discarding the supernatant.
(c) Remove tube from magnet and resuspend the Dynabeads
in 1.0 ml of PBS 2 mM EDTA and 0.1 % BSA.
(d) Repeat step b.
(e) Remove tube from the magnet and resuspend the
Dynabeads in 0.5 ml of PBS 2 mM EDTA and 0.1 % BSA.
3.2.3 Immunomagnetic 1. Add 0.25 ml of the washed Dynabeads to the cell suspension.
Separation 2. Incubate for 5 min at 4 °C with gentle tilting and rotation.
3. Place tube in the magnet for 2 min.
4. Transfer supernatant containing unbound (lineage negative)
cells to a fresh 5 ml polypropylene collection tube.
5. To maximize cell recovery, remove tube from magnet and

resuspend beads/cells in 1 ml buffer.
6. Place tube back in the magnet for 2 min and transfer the super-
natant into the lineage negative collection tube as per step 4.
Discard used beads.
7. Add the second aliquot of washed Dynabeads to the cell sus-
pension in the lineage negative collection tube.
8. Incubate for 10 min at 4 °C with gentle tilting and rotation.
9. Place tube in magnet for 2 min.
10. Transfer the supernatant containing the lineage negative cells
to a new collection tube.
11. Repeat steps 5 and 6.
12. Place lineage negative collection tube in magnet for 2 min
(see Note 11).
13. Transfer the supernatant containing lineage negative cells to a
14 ml polypropylene collection tube.
14. Make up the volume of the lineage negative cell suspension to
10 ml and count.
3.3 Fluorescence 1. Centrifuge lineage negative cells and aspirate supernatant.

Activated Cell Sorting 2. Resuspend cell pellet at 1 × 107 cells/100 μl in optimally
3.3.1 Labeling pre-titered antibody (1 μg/ml anti-mouse CD41-FITC or
for Cell Sorting 0.133 μg/ml CD41-PE) (see Note 12).
3. Incubate in the dark on ice for 20 min.
4. Wash cells in PBS-2 % Se and centrifuge at 300 × g for 5 min,
4 °C.
5. Decant supernatant.
6. Resuspend the cell pellet at 1 × 106 cells/ml in pre-warmed
PBS-2 % Se containing 10 μM Hoechst 33342 (see Note 13).
7. Incubate light protected in a 37 °C water bath for 60 min.
8. Centrifuge at 300 × g for 5 min, 4 °C.
9. Decant supernatant.
10. Resuspend the cells in PBS-2 % Se and filter through a 100 μm
cell strainer prior to cell sorting (see Note 14). A cell concentra-
tion of 1–2 × 107 cells/ml is considered optimal for cell sorting.
3.3.2 Gating Strategies The set up of any flow cytometer, including controls for compen-
for the Isolation of MM sation, is essential for the identification and accurate sorting of spe-
cific subsets of cells. Aliquots of the following cell samples are
required for selection of instrument settings and fluorescence
compensation.
1. Unstained BM cells for setting scatter profiles and gains for
background fluorescence.
Table 1
Flow cytometry instrumentation setup
Fluorochrome label Antibody conjugate Excitation wavelength Optical filter

FITC CD41 488 nm blue 528/19 nm
PE CD41 561 nm yellow 605/20 nm
GFP – 488 nm blue 528/19 nm
RFP – 561 nm yellow 605/20 nm
Hoechst – 355 nm UV 460/25 nm
All parameters are in logarithmic scale
2. BM cells from GFP or RFP mouse for compensation control.

3. BM cells stained with CD41-FITC or -PE for compensation
control.
4. BM cells stained with Hoechst for compensation control.
All signals are measured on a logarithmic scale as specified in
Table 1.
Total MM can be isolated based on the combination of side
scatter (SSC) profile and CD41 expression (SSChighCD41bright);
which account for approximately ~0.2 % of whole BM (Shen man-
uscript submitted). Figure 2a, b shows gating strategy and Fig. 2c,
d shows sorted MM under phase contrast using a light microscope.
Sequential gating strategy for the isolation of MM based on ploidy
is described in Table 2 and Fig. 3a–c (see Notes 15–21). Sorted
MM are shown in Fig. 3d.
3.3.3 Cell Collection Cells are sorted and collected at 4 °C into 1.7 ml microtubes
containing 0.5 ml of PBS-2 % Se (see Note 22). Reanalysis of the
sorted populations is not routinely performed due to the low cell
numbers recovered. Instead, the purity of the sorted populations is
assessed using a hemocytometer and microscope equipped with
phase contrast and fluorescence (see Notes 23–27).
4 Notes
1. Depending on experimental requirements, MM are sorted

from C57Bl/6J, GFP, or RFP mice. Due to the FACS time
required, we routinely process three mice per experiment.
Aside from the fluorophore to which CD41 is conjugated
(FITC for C57Bl/6 or RFP mice and PE for GFP mice), no
other changes are made to the protocol. We often sort MM
from GFP or RFP mice because GFP+ or RFP+ MM are easily
Fig. 2 FACS isolation of viable, pure MM sorted on CD41 expression. (a) Enrichment gate for SSChighCD41bright
MM (R1). (b) Final sort gate for SSChighCD41bright MM post-enrichment (R2). (c) Phase contrast light microscopy
image of FACS sorted SSChighCD41bright MM (20×). (d) FACS sorted MM labeled with CD41 FITC (green), DNA is
shown in blue (40×)
Table 2
Sequential gating strategy and region (R) definitions for sorting MM based on ploidy
Region Gating strategy Mode Target

Sort 1 Enrichment
R1 FSC versus SSC FSChighSCChigh
R2 Hoechst versus CD41 (through R1) Hoechst 8 N and 16 N, CD41+
R3 Hoechst versus CD41 (through R1) Hoechst 32 N and 64 N, CD41+
Sort 2a (from R2) Purity recovery
R4 Hoechst versus CD41 (through R1) Hoechst 8 N, CD41+
Sort 2b (from R3) Purity recovery
All parameters are in logarithmic scale
Fig. 3 Isolation of viable and pure high ploidy MM. (a) All cells are processed through the FSChighSSChigh gate
(R1), during a two-way enrichment sort of 8 N + 16 N MM one-way (R2) and 32 N + 64 N the other way (R3).
To ensure that adequate events can be visualized and the gates are set accurately, R2 and R3 gates are set on
an image that is not gated through R1. (b) All cells are repassed through R1, during the subsequent purity
recovery sort of 8 N MM one-way (R4) and 16 N MM one-way (R5). (c) All cells are repassed through R1, during
the subsequent purity recovery sort of 32 N MM one-way (R6) and 64 N MM one-way (R7). (d) Phase contrast
light microscopy images of sorted high ploidy MM. Hoechst stained DNA is shown in blue (40×)
distinguished from both each other and other cells of

C57Bl/6J origin.
2. This osmolarity results in the maximal recovery of murine cells.
3. This results in the removal of approximately 60 % of unwanted
lineage committed cells. The addition of Ter119 to our lineage
depletion cocktail reduced our MM count by 15.4 %, so it was
excluded.
4. Three different nozzle sizes are available at our FlowCore
Cytometry Facility (Monash University, Melbourne, Australia):
70 μM, 100 μM, or 140 μM. In FACS, the nozzle used to sort
cells should be four to five times the diameter of the target cell
[15]. Due to the size of our target cell, we tested the two larger
nozzle sizes. Despite MM having delicate cytoplasm, because
all cells are buffered as they run through the FACS machine,
MM are able to maneuver through the machine and through
the 100 μm nozzle without shearing. Although results are
comparable when MM are sorted through the 140 μM nozzle,
set at 12 psi and ran at ~2,000 events per second, the sort is
significantly slower and sorting large numbers of MM is not
feasible due to stream instability. Therefore, although not opti-
mal in terms of target cell size in relation to nozzle size, we
sort MM using the 100 μm nozzle.
5. Remove tibia, femur and iliac crest from the spinal cord of the
mouse. Remove the majority of muscle tissue from the three
bones by carefully scraping the bones with the scalpel blade.
Separate the tibia and femur by dislocating the knee and excise
the tibia by pulling the foot and peeling away residue muscle.
Remove the patella and scrape the area around the head of the
femur. To allow a 21 gauge needle to be inserted, shave a small
sliver of bone from behind the femoral head. When the muscle
is removed from the iliac crest, a flat triangular piece of carti-
lage is exposed. Remove this cartilage by cutting through the
acetabular notch [16].
6. Due to the size of MM and their delicate cytoplasm, we pellet
cell suspensions at 300 g.
7. To remove debris and cell clumps following flushing of the
central BM, we filter the cell suspension through a 100 μM
filter. This sized filter is preferred over a 70 μM filter to mini-
mize MM loss.
8. It is a good practice to centrifuge antibodies briefly in a
microfuge before use; the supernatant is then used, eliminating
nonspecific background staining by any protein aggregates
formed during storage.
9. Because B-cells and myeloid cells account for the majority of
cells in the central BM, to reduce the quantity of sample going
onto the sorter and to enrich for MM, these mature lineage
cell types are depleted using the Dynabeads system. The num-
ber of Dynabeads used was optimized based on depletion effi-
ciency and experimental cost.
10. Beads are washed to remove the sodium azide in the bead stor-
age buffer.
11. This step is to ensure the removal of any residual Dynabeads
from the cell suspension.
12. During our experiments, we have found the PE conjugation of
CD41 is much brighter than FITC-CD41 and therefore we
use the two antibodies at different concentrations. Due to
both the size of MM and their high expression of CD41, when
sorting, a logarithmic scale for flurochrome detection is used
with decreased FITC or PE voltage settings. The FITC or PE
voltage is set based on CD41 positive MM being set at the
third decade.
13. We also tested the DNA binding dye Nuclear Red (Draq 5)
which does not require a UV laser. The ploidy populations are
not as distinct as those seen with Hoechst 33342 and due to
the cost of the reagent; we do not use Nuclear Red routinely.
However, if no UV laser is available, this reagent can be substi-
tuted for Hoechst 33342.
14. In order to reduce the incidence of nozzle clogs during sort-
ing, sort as soon as possible after labeling and filter the sample
immediately prior to sorting.
15. Although some FACS machines are able to sort “four-ways,”
due to the large size of the target cells and possible accuracy
issues, we only ever sort MM “two-ways.”
16. When we first attempted to sort MM, our samples were occa-
sionally contaminated by unwanted hemopoietic cells that
would proliferate in culture. Although in theory only large
(Forward Scatter; FSChigh) cells of high granularity (SSChigh)
should be sorted, because CD41 is also expressed on hemopoi-
etic stem and progenitor cells, these cells could be sorted if
they remained as cell clumps or were attached to MM. To opti-
mize purity, we tested a number of before sorting and during
sorting strategies (see Notes 17–19).
17. Prior to sorting, we performed a Nycoprep gradient pre-
lineage depletion. However, we determined that this addi-
tional step reduced the number of MM by 82.7 %.
18. We also tested a red cell lysis with NH4Cl prior to the lineage
depletion step. We demonstrated that this additional step
reduced our MM population by 47.1 %.
19. To maximize MM purity, our lineage depleted central BM
sample was first sorted through the “enrich” mode of the
Cytopeia Influx 516SH. Cells are processed at up to 10,000

events per second. For the final sort, the enriched sample was
sorted on the “purity recovery” mode at 5,000 events per sec-
ond. This mode yielded better purity and cell count accuracy
compared to “purity yield” and “large particle recovery”
modes. Passing the cells through the sorter twice increased
purity; however, MM recovery is severely reduced. Although
highly pure, sorting MM in this manner, results in a 7 % recov-
ery of the estimated total MM population in central BM. Post
double sorting our average yields per mouse are (taken from
49 sorts of the BM from three mice per sort):
(a) 8N: 1756 cells per mouse (12.6 % of sorted MM).
(b) 16N: 9109 cells per mouse (65.4 % of sorted MM).
(c) 32N: 3035 cells per mouse (21.8 % of sorted MM).
(d) 64N: 32 cells per mouse (0.2 % of sorted MM).
These ratios of different MM ploidies are equivalent to
those previously published by Ebbe and Boudreaux [17],
where they used chromophore intensity following Feulgen
staining to measure MM ploidy in BM smears.
20. If total MM are sorted based on CD41 expression, an enrich-
ment sort is performed through a SSChighCD41bright gate and
the purity recovery sort performed through a tighter
SSChighCD41bright gate. If ploidy populations are required, a
two-way enrichment sort is performed collecting 8 N + 16 N
MM one-way and 32 N + 64 N the other way. Single ploidy
populations are then isolated in two subsequent purity recov-
ery sorts (8 N one-way and 16 N one-way, then 32 N one-way
and 64 N one-way).
21. In addition, because in “enrich” mode we sort the samples at
twice the speed of “purity recovery” mode, we are able to pro-
cess more sample in less time. Approximately four hours of
sorter time is required to isolate the four different ploidy pop-
ulations from three mice.
22. MM can also be sorted into 5 ml tubes containing 1 ml of buf-
fer or a 96 well plate containing 50–100 μl of medium.
23. Post-sorting, when manual counts are performed, we consis-
tently obtain approximately 50 % of the sorter MM count. We
do not believe the other 50 % of “MM” in the count are con-
taminating cells because they cannot be visualized under the
microscope and do not proliferate in culture. Random debris
would also be easily seen and due to the gating strategy, only
FSChighSSChigh “cells” should be sorted; thus, small dead cells,
cell fragments, or platelets should not contribute to the count
and should not be sorted. It is possible that MM may be
counted and then die; however, if this occurred, the cell or cell
nucleus should still be visible. The stream is targeted before

each sort so the cells should enter the tube without incidence.
In addition, if there are targeting problems, we would not
expect to consistently find 50 % of the sorter count in our col-
lection tube or plate, we would expect more variability depend-
ing on the person targeting the stream.
24. Immediately after sorting, MM often appear as a “ball” with
the cytoplasm not clearly visible. Following time in culture,
MM spread out and the nucleus becomes clearly visible within
the immense cytoplasm.
25. The purity of different ploidy populations was tested and con-
firmed by RT-PCR for genes that are known to increase in
expression as MM mature [2].
26. We have isolated MM for experiments such as qRT-PCR as
well as for culture studies where we test MM cytokines release
after 1 week [2].
27. Immunohistofluorescence analysis revealed ~60 % of total MM
are located within central BM and ~40 % of total MM are
within 12 cell diameter of the bone–BM interface (the endos-
teal region [2, 18, 19]). In order to isolate MM from the end-
osteal region, we analyzed the incidence of MM following
gentle crushing of cleaned bones in a mortar and pestle.
However, this process resulted in a 69.8 % loss of megakaryo-
cytes and was most likely due to the crushing force damaging
the delicate cytoplasm of MM. Next, we analyzed MM ploidy
distribution following crushing. Although we found that all
ploidy populations were present, crushing and enzyme treat-
ment (collagenase and dispase to release cells attached to the
bone) reduced the incidence of higher ploidy MM. Therefore,
while still possible, the isolation of endosteal MM following
crushing and enzyme treatment does not yield significant
numbers of MM, or maintain the ratio of MM ploidy evident
in unmanipulated BM. In addition, while the sort enrichment
step increases MM purity, it further reduces the yield.
Therefore, sorting endosteal MM appears to be a very time
and resource consuming effort that may not prove useful for
experiments where a large number of pure and viable MM is
required.
Acknowledgments
The authors thank Dani Cardozo for assistance with animal work,
Michael Reitsma and Andrew Fryga for intellectual input and flow
cytometric support. In addition, we also thank Kathryn Flanagan
and Karen Clarke for flow cytometric support.
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Chapter 11
Looking for the Niche: Substance Delivery into the Lateral

Ventricle of the Brain: The Osmotic Minipump System
María Victoria Gómez Gaviro, Pedro Luis Sánchez Fernández,
Robin Lovell Badge, and Francisco Fernández Avilés
Abstract
The infusion of different substances into the left ventricle of the brain allows substances to reach the
subventricular zone, one of the neural stem cell niches in the adult brain. Implantation of an osmotic
minipump delivers proteins, virus and drugs directly into the lateral ventricle to act locally. Here we
describe this method consisting on a cannula implanted into the lateral ventricle and linked to an osmotic
minipump via catheter. The cannula is fixed over the brain and the minipump is placed subcutaneously.
This system can be maintained from days up to several weeks and ensures constant and regular delivery of
the desired biological product.
Key words Brain, Minipump, Skull, Cannula, Stereotaxic frame, Substances administration, Neural
stem cell niche
1 Introduction
There are two neural stem cell niches in the adult mammalian
brain: the subventricular zone (SVZ) and the dentate gyrus in the
hippocampus (DG) [1, 2]. Located on the lateral wall of the lateral
ventricles, the SVZ is composed of different cell populations,
including a monolayer of ependymal cells that lines the ventricle,
NSCs, transit-amplifying cells, neural progenitors (neuroblasts),
astrocytes, and a dense network of blood vessels. The stem cell
niches contain several cell types that produce a range of factors to
control how fast the stem cells divide and the type of cells to which
they give rise.
The brain is not an easily accessible organ to inject substances,
because it is surrounded and protected by the skull. To access the
SVZ, niche it is necessary to get through the skull after calculating
135
136 María Victoria Gómez Gaviro et al.
a b cannula
z
SVZ
y
cb ob
Lateral
ventricles
Fig. 1 Schematic of the brain coordinates to localize different regions and the mouse LV. (a) “y” indicates
rostro-caudal orientation, “z” dorsoventral, and “x” the lateral coordinates. Ob olfactory bulb; Cb, cerebellum;
SVZ, subventricular zone. (b) Schematic of a brain coronal section with the lateral ventricles and SVZ localiza-
tion; it shows how the cannula penetrates into the LV
the coordinates to localize the proper part of the brain to deliver

the substance to. This coordinates are the deepness, the latero-
ventral and rostro-caudal characteristics (Fig. 1) [3].
The osmotic pumps are miniature, infusion pumps for the con-
tinuous dosing of unrestrained laboratory animals as small as mice
and young rats. In case of the brain the minipump is placed in the
intercapsular of the animal and linked to a cannula via a catheter
(Alzet, CA).
This technique has been widely used in the cell therapy and
gene therapy fields [3–6]; different substances including peptides,
growth factors, recombinant proteins, virus, and other biotechnol-
ogy compounds are loaded onto deposited inside the minipump
and are delivered predictably at controlled rates. Recent work has
demonstrated that this technique is useful to study proliferation,
neurogenesis, regeneration, and cell fate among other biological
processes [3–5].
Different studies have been focused on the comparison
between injection and infusion [7–9]. Osmotic minipumps repre-
sent a comfortable method for chronic dosing and have a wide
range of advantages, including continuous administration of short
half-life proteins, avoidance of animal handling and stress, control
of therapeutical concentrations, reduction of side-effects, and
higher efficacy-delivery of substances to a specific target or tissue.
In summary, this is a very well-accepted and widely used
method to administrate substances either systemically or to a spe-
cific organ region or tissue.
Delivery Substances with the Osmotic Minipump 137
2 Materials
2.1 The Osmotic 1. The system is provided with the flow moderator (Alzet
Minipump minipumps 1007D, flow rate 0.5 μl/h). This minipump delivers
substances for 7 days.
2. Brain infusion kit 3 (Alzet): includes the brain infusion can-
nulae, vinyl catheters tubes, and depth-adjustment spacers.
Cannulae in kit number 3 are 3 mm long, so, the cannula pen-
etrates 3 mm under the skull, where the lateral ventricles are.
3. Water bath 37°.
4. NaCl (0.9 %) (Sigma).
5. Forceps, scissors, insulin syringe, and insulin needle.
2.2 Chemicals 1. Analgesics: Fentanyl, Buprenorphine, and Ibuprofen.

and Other Reagents 2. Anesthetic: Sevoflurane or equivalent.
3. NaCl (0.9 %) (Sigma).
4. Ethanol (70 %).
2.3 Surgery 1. Stereotaxic frame.

2. Microscope.
3. Vaporizer.
4. Mask, gloves, Blades and scalpel no 15, gauzes.
5. Small Drill.
6. Scissors and Forceps.
7. Ethanol (70 %), NaCl (0.9 %), Gauzes.
8. Veterinary-grade tissue adhesive (Vetbond, 3 M).
3 Methods
3.1 Preparation of A range of sizes, flow rates and durations are available. The
the Osmotic Minipump minipump should be chosen and according to the needs of the
experiment (the flow rate and volume). Proliferation studies in
the mouse SVZ can be done with Alzet 1007D or Alzet 1003D
minipumps [3].
Prepare the system the day before the surgery using sterile
conditions (in a hood or other sterile cabinet and gloves should
be used).
Load the osmotic minipump (Alzet minipumps 1007D, flow
rate 0.5 μl/h) with the substance avoiding making bubbles.
Incorporate the needle into the minipump with an insulin syringe
(see Note 1). Afterwards, connect the minipump with the flow
moderator.
Flow moderator
catheter
cannula
minipump
salt sleeve
Semi-permeable
membrane
Impermeable
membrane
Suspension, protein…
Fig. 2 Components of the minipump system. The osmotic minipump is composed of the flow moderator and
the minipump itself. The cannula and the catheter must be linked first and the catheter is then connected to
the minipump
The brain infusion kit must be prepared next: in kit number 3

the length of the cannula is 3 mm, meaning that the cannula pen-
etrates 3 mm under the skull where the lateral ventricles are and
spacers are not necessary. If the 5 mm cannula is chosen, spacers
should be now placed in the cannula. Each spacer is 0.5 mm wide
and for the SVZ 3 spacers are needed. Use the surgical glue to
attach the spacers to the cannula and among themselves (optional)
(see Fig. 2). Catheter length should be measured (from back to the
brain) and cut in such a way that around 2 cm are left to be con-
nected to the minipump (see Note 2). Now connect the cannula to
the catheter.
Using forceps place the minipump with the catheter and can-
nula in a 50 mL Falcon tube filled previously with NaCl 0.9 %.
Leave it at least 16 h at 37° in a water bath. This is important to
activate the system: the minipump must be at 37° at the moment
of the implantation to start releasing the content (see Note 3).
3.2 Surgery Prepare the surgical area: stereotaxic frame, mask, vaporizer, O2,
sevoflurane, buprenorphine, fentanyl, surgical instruments.
The mouse will be anesthetized using general anesthesia by
inhalation.
Induction anesthesia should be done using an induction cham-
ber, with 2 % O2 and 6 % sevoflurane. When the animal is asleep
and has no reflex (check the pedal reflex) decrease the dose of
Delivery Substances with the Osmotic Minipump 139
anesthetic to 2–3 % and the O2 to 1–1.5 %. Take the animal out the
box and place it to the stereotactic frame. Put the mask on the
mouth/nose of the animal.
Administer 0.2 μg/ml/g of the analgesic fentanyl (or equiva-
lent) with an intraperitoneal (i.p.) injection. Fix the tail and the
legs to the surface. Spread ethanol 70 % over the head skin. Cut the
skin over the skull and open a hole with a scalpel number 15 on top
of the head (between the eyes and over them) and expose the skull
area (see Note 4). Clean up the surface of the skull to localize the
bregma. Crack the skull softly with a scalpel to remove the menin-
ges and visualize the bregma easily.
With the stereotactic frame measure 0.0 mm relative to bregma,
1.2 mm lateral, and 3.5 mm deep to inject the substance into the
SVZ. Alternatively, locate the position relative to bregma −2.0 mm
posterior, −1.95 mm lateral, and −1.9 mm ventral to the pial sur-
face to inject the substance in the hippocampus (in case of injec-
tions into the DG). Make a small hole with a drill in the area where
the coordinates indicate. This hole will allow you to open enough
space subcutaneously with the forceps to introduce the minipump
in the intercapsular region, without the need to open a new hole
on the back. Put a little amount of glue between the cannula and
the skull, in order to fix it to the skull. Close the skin over the can-
nula and put glue over the skin (see Note 5). Inject Buprenorphine
i.p. Decrease the anesthetic concentration but maintain the oxygen
for post-operative care.
Once the mouse is awake, the osmotic minipump will deliver
the substance directly into the ventricle or the DG homogenously
during a long period of time.
4 Notes
1. This step must be done very carefully and slowly. The mini-
pump will be full when a small drop appears over the top of the
minipump.
2. In the case of an adult MF1 mouse, the length of the catheter
should be around 2 cm. This must be long enough to avoid
the movement of the minipump in the interacapsular space.
3. The minipump should be manipulated with hands as little as
possible; it is advisable to use forceps to handle it.
4. Hair on the top of the brain can be removed with a shaver or
with scissors.
5. If the hole is small it is more convenient to close it with surgical
glue instead of sutures; it is quicker.
Acknowledgments
This worked was supported by a Marie Curie Intra-European

Fellowship to MV.G-G (275885).
References
1. Doetsch F (2003) A niche for adult neural stem 6. Xia HJ, Suda S et al (2011) ACE2-mediated
cells. Curr Op Genet Dev 13:534–550 reduction of oxidative stress in the central nervous
2. Moore KA, Lemischka IR (2006) Stem cells system is associated with improvement of auto-
and their niches. Science 311:1880–1885 nomic function. PLoS One 6(7):U548–U558
3. Gómez-Gaviro MV, Scott C et al (2012) 7. Bedard AM, Maheux J et al (2011) Continuous,
Betacellulin induces proliferación in the neural but not intermittent, antipsychotic drug deliv-
stem cell niche and induces neurogenesis. Proc ery intensifies the pursuit of reward cues.
Natl Acad Sci USA 109(4):1317–1322 Neuropsychopharmacology 36(6):1248–1259
4. Charlotte E, Gómez-Gaviro MV et al (2010) 8. Borkiewicz P, Pawlak M, Mackowiak P (2011)
SOXE proteins, acting downstream of Sonic The results of prolonged action of GLP-1 on
Hedgehog signalling, are required for the initi- some metabolic parameters. Folia Biol (Krakow)
ation and maintenance of neural stem cell prop- 59(1–2):13–17
erties. J Nat Neurosci 13(10):1181–1189 9. Fujisawa T, Nakashima H et al (2011) Targeting
5. Ramírez-Castillejo C, Sánchez-Sánchez F et al IL-13Ralpha2 in human pancreatic ductal
(2006) Pigment epithelium-derived factor is a adenocarcinoma with combination therapy of
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Chapter 12
Unbiased Stereological Method to Assess

Proliferation Throughout the Subependymal Zone
Ana Mendanha Falcão, Joana Almeida Palha, Ana Catarina Ferreira,
Fernanda Marques, Nuno Sousa, and João Carlos Sousa
Abstract
The subependymal zone (SEZ), frequently named as adult subventricular zone (SVZ), is a niche of adult
neural stem and progenitor cells that lines a large extension of the lateral ventricles of the brain. The majority
of the studies do not analyze the SEZ throughout its entire extension. Instead, studies of cell populations
within the SEZ typically focus their analysis on a narrow space between specific bregma coordinates that
provides a perspective of only a small portion of the SEZ. We have previously proposed a standard division
for the SEZ at the anterior–posterior and dorsal–ventral axes based on external brain anatomical hallmarks
(Falcao et al., PLoS One 7:e38647, 2012). Herein, we describe in detail the procedure and a stereological
approach that can be used to obtain an unbiased estimation of the SEZ cell proliferation under physiologi-
cal and pathological conditions. This approach takes into consideration clear SEZ anatomical divisions,
both on the anterior–posterior and dorsal–ventral axes, which will standardize future studies on the SEZ.
Key words Subependymal zone, Adult subventricular zone, Neural progenitor cells, Stereology,
Proliferation, Topography
1 Introduction
In recent years, the number of manuscripts focusing on the subep-

endymal zone (SEZ), also named adult subventricular zone (SVZ),
increased sharply. A substantial portion of these studies relies on
estimations of the SEZ cell proliferation. Recently, a growing body
of evidence indicates that the adult neural stem cells (NSC) and its
progeny are highly variable along the SEZ niche [1, 2]. Thus, the
estimation of the proliferation rates along the SEZ should take into
account these topographic gradients. The estimation of cell prolif-
eration is a valuable tool to analyze the SEZ stem cell niche dynam-
ics under determined conditions; for instance, in response to stroke
and in neurodegenerative diseases as well as in response to specific
stimuli, such as the intraperitoneal or intracerebroventricular
administration of molecules/factors, and in transgenic mice [3–6].
141
142 Ana Mendanha Falcão et al.
There are many different approaches to estimate cell proliferation

in the SEZ, including estimation of total number of proliferating
cells using optical fractionator method [7, 8], proliferating cells per
area [9], per volume [10], or per section [11].
Stereology is, by definition, the three-dimensional interpretation
of planar sections of materials or tissues, and it aims to quantify
properties of 3D objects from serial 2D sections of the sample
[12]. When analyzing SEZ cell proliferation, in order to get an
unbiased stereological methodology, one must follow three key
principles: (1) systematic uniform random sampling, i.e., the first
section to be analyzed is selected randomly, and the subsequent
sections must be apart from each other at consistent intervals,
(2) that the sample has no constant pattern, i.e., there is no repeti-
tive layer patterning in the object under analysis, and (3) the
researcher performing this analysis must be blind to the experi-
mental groups, i.e., the person who is analyzing the sections
should not know the identity of the experimental groups being
examined. After randomly selecting the sections, that may either
comprise the entire SEZ or just its anterior, intermediate, poste-
rior, and post-posterior division (as described in Falcao et al.
[13]), the SEZ area is estimated for each section and the total
number of proliferating cells [5-bromo-2′-deoxyuridine (BrdU)
or Ki67 positive cells] are counted within the drawn area. The
optical fractionator method estimates the total number of cells
(or alternatively proliferating cells) from the number of cells esti-
mated in a systematic randomly sampled set of unbiased virtual
counting spaces covering the entire region of interest, i.e., the
SEZ area previously drawn, with uniform distance between count-
ing spaces in directions X, Y, and Z. The later method is not the
most indicated, and instead every proliferating cell should be
counted, since the proliferative rates within a single SEZ section
are extremely heterogeneous [13] and thus the optical fraction-
ator method could provide estimation with low precision.
Of notice, the SEZ areas indicated above vary according to the
position in the anterior–posterior axis and, consequently, the num-
ber of cells (total and proliferating) also changes according to their
relative position in the SEZ.
2 Materials
2.1 BrdU Preparation The BrdU dose commonly used to assess proliferation in the SEZ,
for Proliferation both for mice and rat is 50 mg/kg. In order to inject twice the
Assessment volume equivalent to the body weight of the rat prepare a solution
of 25 mg/mL of BrdU (Sigma, St. Louis, MO, USA) dissolved in
sterile saline (0.9 % w/v NaCl). For a volume of 10 mL weight
0.25 g of BrdU in an analytical balance and add it to 10 mL of
Unbiased Stereological SEZ Proliferation Analysis 143
sterile saline solution. For mice, prepare a solution of 5 mg/mL

and inject a volume equivalent to ten times the mice weight. For
20 mL of solution weight 0.1 g of BrdU in an analytical balance
and add it to 20 mL of sterile saline solution. In order to easily dis-
solve the BrdU in saline, warm up the solution while mixing it
(see Note 1). Aliquot the BrdU solution and store it at −20 °C
(see Note 2).
2.2 Immuno- 1. 4 % Paraformaldehyde (PFA) in phosphate saline buffer (PBS):

histochemistry 40 g of PFA in 1 L of 0.01 M PBS. The PFA is dissolved by
Solutions warming up and mixing the solution in a magnetic stirrer
(see Note 3). Store at 4 °C.
2. Tris-buffer saline (TBS) 0.05 M: 6 g Tris base, 9 g NaCl in 1 L
of dH2O. Bring to pH to 7.6. Store at 4 °C.
3. TBS-0.2%T: dissolve 2 mL of TritonX-100 in 1 L of TBS.
Store at 4 °C.
4. HCl 2 M: add 66 mL of HCl 37 % to dH2O and make up to
1 L. Store at 4 °C (see Note 4).
5. Citrate buffer 10 mM: dilute 100× in dH2O from stock solu-
tion citrate buffer 1 M (Thermo Scientific, Waltham,
Massachusetts, USA).
6. Hydrogen peroxide (H2O2) 3 %: dilute 10× in TBS from stock
solution (H2O2 30 %).
7. Bovine serum albumin 4 % (BSA) (Sigma): 4 g of BSA in
100 mL of TBS.
8. Tris–HCl 0.05 M: 6 g Tris base in 1 L of dH2O, bring to
pH 7.6. Store at 4 °C.
9. 3,3′-Diaminobenzidine tetrahydrochloride hydrate (DAB)
(Sigma) substrate solution, 0.025 % (w/v) of DAB in 0.15 %
(v/v) of dH2O2: 75 mg of DAB, 1,5 mL of H2O2 30 % in
300 mL of Tris–HCl (see Note 5).
10. Harris Hematoxylin solution (Merck, Frankfurt, Germany),
Ammonia solution 25 % (Merck) (dilute 100× in dH2O to
0.25 %), ethanol 50, 70, 96, 100 %, 100 % Xylene (Sigma), and
Entellan New mounting medium (Merck).
2.3 Immuno- 1. Mouse anti-BrdU (DAKO, Clone Bu20a, DAKO, Spain).

histochemistry 2. Rabbit anti-Ki67 (Ki67 antigen, rabbit polyclonal antibody,
Antibodies Novocastra, UK).
3. Secondary antibody: biotinylated goat Anti-Polyvalent (mouse
and rabbit) and streptavidin peroxidase from UltraVision
detection system (Thermo Scientific).
3 Methods
3.1 BrdU Injection BrdU, an exogenous marker for proliferation, is a thymidine

analogue that is incorporated in the DNA during the S phase. Inject
BrdU (50 mg/kg) intraperitoneally with a 26 or 25G needle. For
rats the volume of injection from the stock solution (25 mg/mL)
is twice the rat weight, for instance a 300 g rat will receive a 600 μl
injection. For mice, the volume of injection is 10× the weight, i.e.,
mice with 20 g will receive 200 μl from the BrdU stock solution
(5 mg/mL) (see Note 6).
3.2 Brain Freezing 1. Upon transcardiac perfusion with saline, collect the brain and
Procedure and Slices place it in a rectangular mold embedded in tissue tek O.C.T.
Collection compound (Thermo Scientific) that provides an appropriate
matrix for cryostat sectioning at −20 °C (see Note 7).
2. Snap frozen the brain by immersing the mold in a recipient
with isopentane and then into liquid nitrogen for a couple of
minutes until it is frozen (see Note 8).
3. Section the brain in a cryostat and collect the sections to Super
Frost plus slides (Menzel-Glazer from Thermo Scientific).
Make 20 μm coronal sections and start collecting all slices as
soon as the ventricle begins. Stop collecting brain sections
when you see large ventral ventricles at the level of the hippo-
campus. The bregma coordinates for the beginning and end of
the SEZ are the following: rat, bregma coordinates 2.28 to
−3.60 mm; mice, 1.18 to −2.06 mm (see Note 9).
4. The methodology to collect the sections is the following: make
series of 8 slides and collect consecutive sections to consecutive
slides, i.e., if you have series of 8 slides, in one slide each brain
section will be 160 μm distant from the subsequent (Fig. 1).
Following this methodology you will obtain slides with repre-
sentative sections at a defined constant distance from each
other (stereological requirement for proliferation assessment).
The sections collected to glass slides should be stored in slide
boxes and frozen at −20 °C (see Note 10).
3.3 Immuno- This procedure is entirely performed at room temperature, unless

histochemistry for otherwise indicated.
BrdU and Ki67
1. Choose representative slides of the SEZ, i.e., one slide of each
series prepared as indicated above (see Note 11).
2. Fixation: fix the tissue in 4 % PFA for 30 min (see Note 12).
3. Wash 3 × 3 min in TBS.
4. Permeabilization: incubate the slides for 10 min in TBS-0.2 % T.
A A A A A A A A
S1 1 S1 2 S1 3 S1 4 S1 5 S1 6 S1 7 S1 8
+ + + + + + +
1 2 3 4 5 6 7 8
9* 10 11 12 13 14 15 16
17* 18 19 20 21 22 23 24
25* 26 27 28 29 30 31 32
A A A A A A A A
S2 1 S2 2 S2 3 S2 4 S2 5 S2 6 S2 7 S2 8
33* 34 35 36 37 38 39 40
n* n+1"
n+8*
Sn
Fig. 1 Schematic representation of methodology used to collect brain sections. Sections are represented in
numbers (from 1 to 40 and then the following consecutive numbers) and are collected following the number
order represented in the figure; thus, 1 is the first brain section collected and 40 represents the 40th brain
section sliced. The distance between consecutive brain sections collected in the same slide (marked with an *
in slide 1 of series 1) is 160 μm (20 μm × 8); marked with a + in slides 1–8 of series 1 are slices collected
consecutively. Because they contain contiguous brain sections, the first slide (1) and the second slide (2) of the
first (S1) series are basically identical. To select representative sections of the SEZ choose the necessary series
of the same slide number (Sn = series n (n≥1) of the slide n (n1≤n≤8))
6. Antigen retrieval: preheat until boiling the 10 mM citrate

buffer in the microwave. Add the slides to the citrate buffer
and leave them immersed at low potency in the microwave for
20 min (see Note 13).
7. Leave sections to cool down under a hood for approximately
15 min.
8. Immerse rapidly through dH2O.
9. Acidification (this step is only required for BrdU staining):
incubate sections in HCl 2 M for 30 min (see Note 14).
10. Wash 3 × 3 min and 1 × 10 min in TBS.
11. Endogenous peroxidases inactivation: incubate sections for
10 min in H2O2 3 % (see Note 15).
13. Block against nonspecific binding: incubate sections in BSA
4 % for 30 min.
14. Pour the excess of BSA in the slides and place it in a flat
humidified chamber. Add the primary antibody (usually 300 μl
for slide) and incubate overnight at 4 °C. For BrdU (Dako) a
dilution of 1:50 in TBS is used, while for Ki67 (Novocastra)

the dilution is 1:100 in TBS (see Note 16).
16. Incubate in secondary antibody (Thermo Scientific) for
18. Incubate in streptavidin peroxidase (Thermo Scientific) for
19. Wash 2 × 3 min in TBS and 1 × 3 min in Tris–HCl.
20. Develop the reaction in DAB substrate while observing
random sections in the microscope (see Note 19).
21. Counterstain the slides for 5 s in hematoxylin for staining tis-
sue nuclei in blue (see Note 20).
22. Pass through dH2O and then ammonia solution 0.25 %.
23. Dehydrate the tissue through serial passages in increasing
alcohol gradients: 3 min in ethanol 50, 70, 96, and 100 % and
finally in xylene (see Note 21).
24. Using the mounting medium Entellan cover the brain sections
with a coverslip. Wait for 1–2 days until they are dried to start
the microscope analysis (see Note 22).
3.4 Proliferation Sampling methodology: The most important rule in this part is to
Assessment perform a systematic uniform sampling, i.e., to analyze sections at
Throughout the SEZ constant distance intervals, for instance 160 μm or 320 μm (or
even more or less). Using the methodology described above to col-
lect sections in the cryostat, if the analysis is performed for every
section in one slide the SEZ brain sections will be separated by
160 μm intervals; if the analysis is performed for every other sec-
tion in a slide, the brain sections will have 320 μm intervals between
them (see Note 23).
Random selection of first section: the first section to be analyzed is
the one displaying a well-defined juxtaposed ependymal layer
Microscope and software: To estimate the cell proliferation rates
throughout the SEZ use, for example, the Visiopharm Integrator
system (VIS) software in an Olympus BX51 microscope (Olympus,
Hamburg, Germany) or similar software. By using this software
you can draw the areas of interest and count within these areas the
nuclei stained for BrdU or Ki67 (in brown) with a count tool.
Delimitate the SEZ areas at low magnification (40×) and perform
the counting of BrdU positive cells within this defined areas at
high magnification (400×) (see Note 25).
Identification of the different SEZ divisions and regions: The coronal
sections collected in the cryostat comprise SEZ between bregma
Fig. 2 Detail of the anterior subependymal zone displaying the ependymal layer
juxtaposed (arrows) and with BrdU positive cells stained in brown
coordinates 2.28 and −3.60 mm in rat, and 1.18 to −2.06 mm for

mice [14, 15]. Within these coordinates are the anterior, interme-
diate, posterior and post-posterior SEZ (Fig. 3). Table 1 summa-
rizes the divisions of SEZ and the external references used to define
it for both rat and mouse. If the analysis is to be performed in dif-
ferent regions of the SEZ in the dorsal–ventral axis, see Fig. 3 for
detail in area delimitation. Briefly, the anterior SEZ comprises the
beginning of the genu of the corpus callosum where a well-defined
ependymal layer is observed and prolongs to the end of the genu
of the corpus callosum. The intermediate SEZ extends up to the
decussation of the anterior commissure; the posterior SEZ ends at
the beginning of the hippocampus. The post-posterior division of
the SEZ is at the level of the hippocampus and finishes with the
fusion of the dorsal and ventral parts of the lateral ventricles. From
this position on, sparse proliferating cells are detected in the SEZ.
Dorsal–ventral axis regionalization comprises dorsal SEZ, located
in the upper part of the lateral ventricles; the beginning of the
RMS, at the dorsal corner of the lateral wall, the dorsolateral and
the ventral SEZ that are the result of the split of the lateral wall in
two parts: the ventral SEZ is perpendicular to the corpus callosum,
and the dorsolateral SEZ begins at the corner of the lateral wall
and extends up to the ventral SEZ where the lateral wall starts to
direct to the ventral tip (see Note 26).
Data processing: The proliferation rates are estimated as the num-
ber of BrdU positive cells per area (in mm2 or μm2). The data
analysis of the SEZ is dependent on the divisions and/or regions
assessed. For instance, if the goal is to obtain total proliferation in
the intermediate SEZ, determine the rates of proliferation for each
section at intermediate SEZ (i.e., the total number of BrdU posi-
tive cells divided by the total area) and then average all the sections
cc
DG
LV
aca ac
A I P PP
Bregma
A I P PP
undefined RMS dorsal dorsolateral ventral

Fig. 3 Representation of the subependymal zone divisions defined at the anterior–posterior and dorsal–ventral
axes. In the upper panel four anterior to posterior divisions are defined according to the SEZ anatomical
heterogeneity along the neuraxis: anterior (A), intermediate (I), posterior (P), and post-posterior (PP). For the
established divisions, regions are further defined in a dorsal to ventral SEZ orientation, as outlined in the
colored traces (middle panel): rostral migratory stream (RMS; red trace), dorsal (blue trace), dorsolateral
(orange trace), and ventral (green trace). In the anterior division of the SEZ, the area containing proliferating
cells that cannot be defined as RMS is designated undefined (black trace). In the post-posterior division of the
SEZ, few proliferating cells are found lining the ventricle wall and therefore no dorsal–ventral region is defined
(ventricle walls outlined in grey). The images are from the rat brain. ac, anterior commissure; aca, anterior
commissure, anterior part; cc, corpus callosum; DG, dentate gyrus; LV, lateral ventricle. This figure is adapted
from [13] under a CC license
Table 1
Anterior–posterior axis anatomical references for the mouse and rat SEZ divisions
Bregma Bregma
coordinates coordinates
SEZ mouse (mm) rat (mm) Anatomical references
Anterior [1.18; 0.74] [2.28; 1.44] From the beginning to the end of the genu
of the corpus callosum
Intermediate [0.74; −0.14] [1.44; −0.12] From the end of genu of the corpus callosum
to the decussation of anterior commissure
Posterior [−0.14; −0.94] [−0.12; −1.72] From the decussation of anterior commissure
to the beginning of the hippocampus
Post posterior [−0.94; −1.94] [−1.70; −3.60] From the beginning of the hippocampus to
the fusion of the dorsal and ventral parts
of the lateral ventricle
Bregma coordinates are according to Paxinos and Franklin [15] for mice and Paxinos and Watson [14] for rat
analyzed for one animal. Repeat this procedure for all animals.
Group animals into different experimental conditions and calculate
the mean proliferation rate for the group by averaging the prolif-
eration rates of animals within the same group. If, within the inter-
mediate SEZ there is the need to distinguish between regions, i.e.,
RMS, dorsal, ventral and dorsolateral, proceed as mentioned
above. Estimate the BrdU positive cells for each region and the
correspondent areas for every section, average rates obtained for
each section to obtain the proliferation rate of one animal. Repeat
this procedure for all animals and estimate the mean proliferation
rate of a determined region for the group. The same rationale is
applied to estimate every specific division and/or region of the
SEZ (see Note 27).
Statistical analysis: Data can be presented as the mean (±SEM) and
analyzed with any statistical package software such as GraphPad
PRISM 5 software (GraphPad Software Inc., San Diego, CA). The
analysis consists of one-way analysis of variance (ANOVA) with
Bonferroni multiple comparison post-test analysis for single-factor
multiple group comparisons to determine differences between
three or more groups.
4 Notes
1. At lower temperatures this solution often precipitates there-

fore before injecting it to animals confirm that there is no
BrdU precipitated. If there is, slightly warm up the solution
and dissolve it again.
2. Avoid freezing and thawing, preferably use always fresh. After

thawing you will have to resuspend the precipitate by warming
up the solution.
3. Prepare this solution 1 day in advance, it will take some time
to dissolve the PFA and afterwards it has to cool down.
4. Preferably use it freshly prepared.
5. Use lab coat, mask and gloves when preparing and using DAB
solution, since DAB is carcinogenic. Wrap the solution in alu-
minum foil to protect from light. Prepare just prior to usage.
6. Animals should be handled for 1 week before the injections in
order to minimize the stress-induced changes in the
hypothalamus-pituitary axis.
7. Place the brains in the mold oriented in such a position that
later it will be ready to glue in the cryostat holder to make
coronal sections. The orientation of the brain in the cryostat is
crucial to have perfect coronal sections. If the sections are not
strictly cut always in the same orientation it can result in diver-
gent areas for the same SEZ position, being assessed between
different animals.
8. Isopentane is highly volatile and harmful; this step must be
performed in the hood. The time spent in isopentane should
be optimized according to the size of the brain, i.e., rat and
mice brains take different times to be completely frozen. Store
the brains at −20 °C until sectioning.
9. If you are not certain where the ventricles begin, to be on the
safe side you should start collecting before you see the ventri-
cles, for instance as soon as you identify the corpus callosum.
Use the Rat or Mouse Atlas from Paxinos [14, 15] to identify
the main structures of the brain while sectioning.
10. The immunohistochemistry should be performed shortly after
the sectioning. Long periods of storage lead to tissue damage
and antigen loss. These sections are not prefixed in PFA and
therefore are more susceptible to degradation.
11. If you choose slide number one of series 1, you should choose
slide number 1 of series 2, therefore sections are apart from
each other at a constant distance.
12. Since PFA is harmful this step should be performed under the
hood.
13. Place the slides into a slide holder and then dive it into a plastic
recipient, suitable for microwave, filled with citrate buffer.
After 10 min in the microwave, observe if the tissue is not
damaged or detaching from the slides during this procedure
(this happens if the temperature is too high or if the glass slides
used are not appropriate - glass slides should be of the super-
frost type).
14. This step allows the linearization of the DNA strands were the
BrdU is inserted. If antigen retrieval is not performed prior to
this step, 30 min in HCl is not enough to detect BrdU stain-
ing; instead 1 h in HCl will work, however it may result in
nuclear damage, which will difficult the analysis under the
microscope. Use fresh HCl 2 M and always wear gloves.
15. This step is required to avoid nonspecific staining when devel-
oping the immunohistochemistry, because external horserad-
ish peroxidase (HPR) coupled to streptavidin is added to the
tissue and will bind to the biotinylated secondary antibody.
16. Verify if (a) the chamber is humid so your antibody solution
won’t evaporate and (b) the slides are not leaning and there-
fore the antibody is equally distributed.
17. This antibody can be reused once.
18. Streptavidin can be reused once.
19. Observing in the microscope while development occurs will
allow determining the time necessary to see strong brown
staining without background, it may vary between 2 and
10 min.
20. Hematoxylin diluted 4× provides a weaker staining and makes
it easier to observe the BrdU nuclear brown staining.
21. The slides can be kept in xylene for some minutes until sec-
tions are covered with a coverslip.
22. Be careful not to introduce air bubbles between coverslip and
sections.
23. Notice that shorter distance intervals will provide you more
accurate estimation but will increase the time you will spend
on the analysis. Intervals of 160 μm or 320 μm between ana-
lyzed sections typically provide accurate estimations for SEZ
proliferation analysis.
24. The first section analyzed must be assigned randomly (stereol-
ogy principle). In fact the process is already random because it
is not known which slide has the first section comprising the
beginning of the SEZ. This would only be possible if every
section collected would be stained prior to the selection of
slides. If you perfuse animals with PFA (and not only saline as
described herein) the first section is likely to have the ependy-
mal layer not juxtaposed but instead a slightly opened ventricle
can be observed. Furthermore, the areas estimated for the
SEZ will be inferior due to the shrinkage of the brain caused
by PFA perfusion and by the histological procedures.
25. Alternatively, if you perform all the protocol with fluorescence
immunohistochemistry you can do the same analysis by taking
images, in a fluorescence microscope or confocal microscope,
of the entire SEZ and then estimate the areas and the cell
counting numbers in the image J software or in the software

provided by the confocal manufacturer.
26. If proliferation is not to be assessed in the entire SEZ, the
division(s) of interest to analyze can be selected by following
the criteria in Table 1. Most studies on the SEZ focus on the
intermediate and the posterior SEZ.
27. To estimate not only the total proliferation but also the prolif-
eration rates in the different regions draw the areas of interest
and count the number of BrdU positive cells within those
areas, independently. Then, to obtain total area and total
BrdU cells, sum the values for all regions, i.e., total = RMS +
dorsal + dorsolateral + ventral.
References
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Biol 73:357–365 ischemia. Neuroscience 105:33–41
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(2003) Neurogenesis and aging: FGF-2 and zone. BMC Neurosci 9:117
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Chapter 13
Cardiac Stem Cell Niche, MMP9, and Culture

and Differentiation of Embryonic Stem Cells
Paras Kumar Mishra, Nicholas John Kuypers, Shree Ram Singh,
Noel Diaz Leiberh, Vishalakshi Chavali, and Suresh C. Tyagi
Abstract
Embryonic stem cells (ESC) are totipotent, self-renewing, and clonogenic, having potential to differenti-
ate into a wide variety of cell types. Due to regenerative capability, it has tremendous potential for treating
myocardial infarction (death of myocardial tissue) and type 1 diabetes (death of pancreatic beta cells).
Understanding the components regulating ESC differentiation is the key to unlock the regenerative
potential of ESC-based therapies. Both the stiffness of extracellular matrix (ECM) and surrounding niche/
microenvironment play pivotal roles in ESC differentiation. Matrix metalloproteinase-9 (MMP9) induces
fibrosis that causes stiffness of the ECM and impairs differentiation of cardiac stem cells into cardiomyo-
cytes. Here, we describe the method of ESC culture and differentiation, and the expression of MMP9 and
its inhibitor, tissue inhibitor of metalloproteinase-4 (TIMP4) in differentiating ESC.
Key words Stem cell, MMP9, TIMP-4, Differentiation, Extracellular matrix, Cardiomyocytes
1 Introduction
Stem cell therapy is an emerging area in cardiovascular diseases,

where the dead myocardium after myocardial infarction (MI) can
be replenished by stem cell transplantation [1–8]. However, the
mechanism of stem cell mediated regeneration of myocardium is
still nebulous. Recent studies suggest that the surrounding niche
plays pivotal role in stem cell differentiation [6, 9–17]. One of the
key factors involved in survival and differentiation of stem cell is
mechano-sensitivity of surrounding niche, which is provided by
stiffness of extracellular matrix (ECM) [18–21]. The stiffness of
ECM determines the contractility of cardiomyocytes and cardiac
tissue repair [22, 23]. One of the important enzymes involved in
induction of stiffness of ECM is matrix metalloproteinase-9
(MMP9) that induces cardiac fibrosis [24, 25]. Although clinical
studies revealed that MMP9 plays crucial role in diastolic
153
154 Paras Kumar Mishra et al.
dysfunction [26], protects against ischemia–reperfusion [27], and

acts as biomarker for heart failure [28], its role in stem cell differ-
entiation was not clear. Recently, we have reported that MMP9 is
involved in regulation of survival and differentiation of cardiac
stem cells by inducing stiffness of ECM [25]. Since ablation of
MMP9 differentially regulate miRNAs [29] and miRNAs play key
role in differentiation of stem cells [8, 30–34], it is also expected
that MMP9 may inhibit differentiation of stem cells by regulating
miRNAs. However, role of MMP9 in embryonic stem cells (ESC)
differentiation is unclear. Therefore, we determined the expres-
sions of MMP9 and its inhibitor, tissue inhibitor of metalloprotein-
ase-4 (TIMP4) in differentiating embryonic stem cells.
We cultured disaggregated mouse embryonic stem cells (ESC)
without mouse leukemia inhibitory factor (LIF) and allowed it to
differentiate. Since heart is the first organ formed during embryo-
genesis [35], we expected to obtain cardiomyocytes, the default
pathway of differentiation. We determined the levels of MMP9 and
TIMP4 in both differentiated and undifferentiated ESC.
2 Materials
Use all the materials in sterile conditions. The ESC and mouse
embryonic fibroblast (MEF) cells should be stored in a liquid
nitrogen tank, whereas the culture medium should be stored
at 4 °C.
2.1 Mouse ESC 1. Prepare Dulbecco’s Modified Eagle Medium (DMEM)

Culture Medium medium by dissolving DMEM powder with high glucose,
Components L-glutamine, pyridoxine hydrochloride, and 110 mg/L sodium
pyruvate but without NaHCO3 into doubled distilled water at
15–30 °C with gentle stirring. The amount of water should be
5 % less than the total desired volume. Add 3.7 g of NaHCO3/l
(Gibco) into the medium.
2. Adjust pH to 7.1–7.2, which is 0.2–0.3 below the desired pH
7.4. The container should be closed after pH adjustment. The
pH rises 0.1–0.3 upon filtration.
3. To prepare complete medium, add 1 % Nonessential amino
acid (Gibco), 1 % sodium pyruvate (Gibco), 20 % fetal bovine
serum (FBS) (Atlanta Biologica), 1 % Penicillin/Streptomycin
antibiotics (Cellgro), and 0.1 % β-Mercaptoethanol (Gibco)
into the DMEM medium. To prepare 609.2 ml of complete
medium, add 6 ml of nonessential amino acid, 6 ml of sodium
pyruvate, 120 ml of FBS, 1.2 ml of 50 mm β-Mercaptoethanol,
6 ml of antibiotics into 470 ml of DMEM medium.
4. Add mouse leukemia inhibitory factor (LIF) (Millipore) at the
concentration of 125 U/ml to the ESC culture medium
(see Note 1).
Cardiac Stem Cell Niche, MMP9, and Culture and Differentiation… 155
5. Immediately sterilize the above medium by passing it through

a 0.22 μm polyethersulfone sterilizing filter (Corning) inside
the Biological safety cabinet (see Note 2).
6. Store the complete sterilized medium at 4 °C.
7. Warm the medium to 37 °C before using it for culturing.
2.2 ESC 1. ESC culture medium without LIF.

Differentiation
Medium Components
2.3 MEF Culture 1. The MEF culture medium is similar to the ESC culture medium
Medium Components except the concentration of FBS is 10 %.
2.4 MEF Inactivation 1. MEFs are frozen in MEF culture medium + 10 % DMSO at
5 × 106/cryovial and stored in liquid nitrogen. MEFs can be
inactivated by γ-irradiation (5,000 rads) in a cell irradiator
packed with dry ice to prevent thawing during irradiation.
They are used as a feeder layer for ESCs (see Note 3).
2.5 Additional 1. Ultralow attachment 6-well plate (Costar).

Components 2. Sterile tissue culture dish (10 cm)
3. Ultrapure water with 0.1 % gelatin (Millipore).
4. Light sensitive and without phenol red, TrypLE Express
trypsin (Gibco).
5. Cell culture grade Dimethyl sulfoxide (DMSO) (Fisher
Scientific).
6. Ultrapure distilled water (Gibco).
7. Dulbecco’s Phosphate Buffered Saline (DPBS) (Gibco).
3 Methods
3.1 MEF Culture 1. Thaw frozen MEFs quickly in a water bath maintained at 37 °C
for 90 s and dilute vial contents with MEFs medium in a 15 ml
conical tube and spin at 270 × g for 5 min (see Note 3).
2. One thawed cryovial of 5 × 106 MEFs is enough for 1 confluent
10 cm plate.
3. Culture γ-irradiated MEFs on a sterile tissue culture plate
coated with 0.1 % gelatin in ultrapure water under a biosafety
cabinet and place in a cell culture incubator maintained at
37 °C with 5 % CO2 (see Note 4).
4. Remove gelatin at the time of ESC plating (see Note 5).
5. To plate MEFs, resuspend the pelleted MEFs in 10 ml MEF
medium, distribute into the gelatin coated plate, and place in
incubator for at least 24 h to allow MEFs to become confluent
before adding ESCs.
Fig. 1 The different stages of embryoid body (EB). (a) One day after plating of embryonic stem cell (ESC) on
mouse embryonic fibroblasts (MEFs) shown in low magnification (10×). The round shape EB is shown by
arrows. (b) The 3 days EB shown in high magnification (20×). (c) The MEF free undifferentiated floating EB.
(d) Differentiated and attached EB
3.2 ESC Culture 1. Twenty-four hours after plating MEFs, remove the medium.
Wash 1× with DPBS. Add 10 ml ESC medium with LIF. Place
back into the incubator for 1 h.
2. Thaw frozen ESCs quickly in a water bath maintained at 37 °C
for 90 s and dilute cryovial contents with ESC medium into a
15 ml conical tube (see Note 3).
3. Spin the tube at 270 × g for 5 min.
4. Remove the supernatant by vacuum using sterilized pipette.
5. Add 5 ml of fresh warm ESC medium with LIF and resuspend
the pellet gently (see Note 6).
6. Distribute this 5 ml of ESC suspension to the 10 cm plate
containing the MEFs and place back into the incubator
(see Note 7).
7. The plated ESCs exhibit a small round morphology. They
attach to the MEFs and proliferate forming oval shaped colo-
nies (Fig. 1a).
8. Change culture medium every day to allow proper growth of

ESCs. LIF must be added each time during medium change.
9. ESCs can be expanded as needed. Trypsinize the entire plate
for 5 min. Collect trypsinized cells into a 15 ml conical tube
and add an equal amount of ESC medium to counteract the
trypsin. Now proceed from step 3 passaging the ESCs onto as
many MEF feeder cell plates as needed (see Note 8).
10. The aggregate of ESC is called Embryoid body (EB). Do not
allow EB to come into contact with one another as this will
induce differentiation. Undifferentiated EB colonies will have
defined borders, which become less defined when overgrown.
11. When EB attains substantial size (Fig. 1b), passage them or
proceed with ESC differentiation.
3.3 ESC 1. Use ESC medium without LIF for differentiation of ESC (ESC
Differentiation differentiation medium).
2. To remove feeder cells from ESC, coat a 10 cm plate with
0.1 % gelatin for 30–45 min.
3. Remove gelatin at the time of ESC plating (see Note 5).
4. Trypsinized ESCs with MEFs with 5 ml of TrypLE Express
and disaggregate it with a fire polished pipette.
5. Transfer the disaggregated ESC into a 15 ml tube.
6. Add 8 ml of ESC medium to the 5 ml of trypsinized cells and
mix it by pipetting up and down.
7. Centrifuge the cell containing medium at 270 × g for 5 min.
8. Remove the supernatant by vacuum using sterilized Pasteur
pipette.
9. Resuspend the pellet with 10 ml of fresh medium.
10. Transfer the medium into 0.1 % gelatin coated plate and incu-
bate it for 1 h (see Note 9).
11. After 1 h, transfer the medium from 10 cm gelatin coated plate
to a 15 ml tube.
12. Centrifuged the tube at 270 × g for 5 min.
13. Remove the supernatant and resuspend the pellet in 1 ml of
ESC medium.
14. Count the ESC number (see Note 10).
15. Dilute ESCs in a manner that 540,000 ESCs are suspended in
36 ml of ESC medium without LIF (15,000 cells/ml). This
volume is good for two 6-well plates at 3 ml of cell suspension/
well of the plate (see Note 11).
16. The plated cells are not disturbed for 48 h. Differentiating
ESCs will begin to agglomerate into free-floating EBs.
Fig. 2 Different regions of differentiated ESC. (a) From central (black spot, arrow ) to the remote (distant from
the black spot ) of the differentiated EB at low magnification (10×). (b) The high magnification (20×) view of
the area of differentiated ESC, where contractile cardiomyocytes are observed
17. After 48 h, change the medium daily (see Note 12).

18. EB attains considerable size by 72–96 h (Fig. 1c) and are ready
for further differentiation. By default, each EB will differenti-
ate into cardiomyocytes (Fig. 1d). The beating of cardiomyo-
cytes can be observed under light microscope at 10× and 20×
magnifications (Fig. 2a, b).
To determine the expressions of MMP9 and TIMP4 in differ-
entiation of ESC, we stained the differentiated and undifferenti-
ated ESC with MMP9. The results revealed that MMP9 is robust
in undifferentiated (Fig. 3c) than differentiated (Fig. 3a, b) ESC.
We also compared central versus remote regions of differentiating
ESC for MMP9 expression because the central region is in active
stage of differentiation and remote region has terminally differen-
tiated cells. The results show that MMP9 is down regulated in
central region (Fig. 3a) but comparatively highly expressed in the
remote region (Fig. 3b). Since TIMP4 inhibits MMP9, we stained
the differentiating ESC with TIMP4. The comparison of MMP9
and TIMP4 in central region of ESC revealed that TIMP4 is highly
expressed (Fig. 4) in differentiating ESC whereas MMP9 is attenu-
ated in the same region (Fig. 3a). These findings indicate that
MMP9 is inhibited and TIMP4 is induced during differentiation
of ESC.
In diabetic condition, MMP9 is activated [24, 25]. To under-
stand the effect of hyperglycemia on differentiation of ESC, we
treated differentiating ESC with 5 mm (physiological dose: CT)
and 25 mM (high dose) of D-glucose for 24 h. Both of them are
stained with MMP9. The comparative results revealed that MMP9
is down regulated in CT (Fig. 5a) but it is robust in hyperglycemic
Fig. 3 Expression of MMP9 in differentiating ESC. (a) The expression of MMP9 (green color ) in differentiating
area, the central region of EB. The blue color is DAPI, which stains nucleus. (b) The expression of MMP9 in the
terminally differentiated region (remote from the center of EB). (c) The expression of MMP9 in undifferentiated
EB. Scale bar is 50 µm
Fig. 4 The expression of TIMP4 (green color) in differentiating region of EB. The left panel show DAPI (blue) that
stains nucleus, the middle panel show TIMP4 staining (green) and the right panel show merged imaged with
blue and green. Scale bar is 50 µm
Fig. 5 The differentiated EBs are treated with 5 and 25 mM of D-glucose for 24 h and stained with MMP9
(green) and DAPI (blue). (a) The left panel control (5 mM) group show less expression of MMP9. (b) The right
two panels (b) (i), and (b) (ii) show robust expression of MMP9 (green). Scale bar is 50 µm
ESC (Fig. 5b(i) and (ii)). It suggests that hyperglycemia induces

MMP9 in differentiating ESC that could have inhibitory effect on
differentiation.
4 Notes
1. LIF inhibits differentiation of ESC. Dilute LIF in the ESC

medium and aliquot into small tubes to avoid freeze-thaw
cycle. We used PCR tubes to aliquot in the manner that one
tube can be used in one time.
2. The commercially available complete culture medium can be
also used.
3. During warming of frozen tubes, try to keep the cap region
above the water level. Also, spray 70 % alcohol and clean it with
Kimwipes before taking it into biosafety cabinet. It will help to
keep the cells in sterilized condition.
4. The non-irradiated MEFs can be treated with Mitomycin C at
the concentration of 10 μg/ml for 3–4 h to mitotically inacti-
vate MEFs. Mitomycin C inhibits DNA synthesis and nuclear
division.
5. The removal of gelatin from the plate immediately before ESC plat-
ing provides better coating than removal of gelatin beforehand.
6. We found that adding first 100–200 μL of medium to the pellet

and disaggregating it by pipetting it up and down and then
adding the rest volume of medium into the disaggregated
100–200 μL of medium is better method to dissolve the
pellet.
7. To get homogenous cell suspension, it is recommended to
dispense the thawed cells drop-wise in all parts of the 10 cm
plates.
8. The number of EB in the medium is important because EB has
tendency to attach to each other in vicinity and form a single
big EB. To maintain a moderate and size of EB, it is necessary
to maintain them in a manner that they do not contact.
9. MEF has more adherent capacity and they have higher binding
affinity to gelatin than the ESCs. MEFs are also larger than
ESCs and sink to the bottom of the plate faster than ESCs.
Therefore, majority of MEFs attach to the surface in 1 h
whereas ESCs remain in suspension.
10. The cells number can be counted by hemocytometer. For that,
take 10 μL of cell suspension, and add 90 μL of Trypan blue.
Mix it well and spread 10 μL of mixed solution onto hemocy-
tometer. Score the number of cells in the four quadrants in the
four corners. The number of cells will be calculated by the
formula: Number = (total cells in four quadrants/4) × 10 × 104.
11. To get 100 % confluent MEFs, regular MEFs from two 10 cm
plates are stored in a single vial. These vials are irradiated with
γ-rays and stored in liquid nitrogen. When used for plating
even after cell death during the process of freezing and thaw-
ing, the number of MEFs is high enough for 100 % conflu-
ence. The excess number of MEF does not affect the ESC
binding and culture.
12. To change the medium, collect EBs and medium into a 15 ml
conical tube and allow 2 min for EBs to sink to the bottom.
Aspirate the supernatant without disturbing the settled EBs.
Add 3 ml of fresh ESC medium without LIF. Triturate very
gently to redistribute the EBs without breaking them apart.
The EBs can now be transferred back to the ultralow attach-
ment 6-well plates.
Acknowledgments
This work was partly supported by American Heart Association

grant 11BGIA 7690055 and National Institute of Health grant
HL113281 to P.K.M, and National Institute of Health grants
HL-108621 and HL-74185 to S.T.
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Chapter 14
Human and Murine Skeletal Muscle Reserve Cells

Rana Abou-Khalil, Fabien Le Grand, and Bénédicte Chazaud
Abstract
Study of stem cell phenotype and functions requires their proper isolation. Stem cells isolated from skeletal
muscle are a useful tool to explore molecular pathways involved in the regulation of myogenesis. Among
progenitor cells, a subset of cells, called reserve cells, has been identified, in vitro, in myogenic cell cultures.
This subset of cells remains undifferentiated while the main population of progenitor cells commits to
terminal myogenic differentiation. When replated, these reserve cells grow as new colonies of progenitors.
At the time of differentiation, they reform both differentiated myotubes and undifferentiated reserve cells.
Here, we present a protocol to obtain and further isolate reserve cells from both human and murine
myogenic cell cultures, together with techniques to analyze their cell cycle status.
Key words Skeletal muscle, Stem cells, Satellite cells, Myogenic progenitor cells, Myogenesis, Human,
Mouse, Cell cycle, Nocodazole synchronization, Reserve cells
1 Introduction
Skeletal muscle stem cells, also called satellite cells, reside under the
basal lamina of the myofibers and are normally quiescent. Following
damage to the myofiber, these stem cells proliferate and differenti-
ate to form new myofibers, and self-renew to reconstitute the res-
ervoir of quiescent satellite cells [1–3]. Satellite cells may be
isolated from both human and murine muscle, and give rise to
myogenic precursor cells in vitro. Myogenic cell cultures have been
used for a long time to parallel in vivo experiments and to under-
stand cellular and molecular mechanisms of myogenesis [4, 5].
A subpopulation of quiescent, noncycling, undifferentiated cells has
been identified in myogenic precursor cell cultures. When replated,
these cells, named “reserve cells” (RCs), give rise to cultures that
eventually form both differentiated myotubes and new RCs
[6–11]. RCs, that are in vitro capable of both self-renewal and
myogenic differentiation may be related to satellite cells described
in vivo, responsible for skeletal muscle growth and regeneration.
165
166 Rana Abou-Khalil et al.
Study of these cells allows analyzing the molecular events occurring

at the time of fate choice, i.e., when proliferating progenitors
decide between committing to terminal myogenic differentiation
or reentering quiescence under an undifferentiated state to replen-
ish the pool of stem cells [12–14]. Here, we present a technique to
isolate RCs from primary myogenic cell cultures from both human
and mouse, based on our own experience and previous descrip-
tions [6, 15]. We also propose a method to synchronize human
myogenic precursor cells, useful to address cell cycle issues. Finally,
we set up a method to analyze human myogenic cell cycle by
differentially staining RNA and DNA contents using Pyronin Y
and Hoechst 33342 dyes. Determination of RNA contents allows
distinguishing G0 (quiescent cells, RCs) versus G1 cells (cycling
cells) as progression in cell cycle is paralleled by further increase
in RNA contents [16, 17].
2 Materials
2.1 Materials 1. 50 ml conical centrifuge tubes (BD Biosciences, Franklin

for Isolation Lakes, NJ, USA).
of Human RCs 2. 40 μm cell strainer (BD Biosciences).
3. 100 mm petri dish (BD Biosciences).
4. Growth medium: Ham-F12 supplemented with 15 % v/v
heat-inactivated Fetal Bovine Serum, 100 IU/ml Penicillin
and 100 μg/ml Streptomycin (Gibco Life Technologies,
Grand Island, NY, USA).
5. Differentiation medium: Ham-F12 supplemented with 5 % v/v
and 100 μg/ml Streptomycin (Gibco Life Technologies).
6. Recombinant human basic Fibroblast growth Factor (bFGF)
(Promega, Madison, WI, USA): stock solution at 100 μg/ml
in sterile water.
7. PBS (1×) Ca2+ Mg2+ free (Gibco Life Technologies).
8. HBSS (1×) Ca2+ Mg2+ free (Gibco Life Technologies).
9. Trypsin-EDTA 0.25 % (Gibco Life Technologies).
10. Trypsin-EDTA 0.15 %: dilute Trypsin-EDTA 0.25 % in
HBSS (1×).
11. Primary Human skeletal myogenic cells prepared as previously
described in Chazaud et al. [18].
2.2 Materials for 1. 100 mm petri dish (BD Biosciences).

of Human Myogenic 2. 50 ml conical centrifuge tubes (BD Biosciences).
Cell Synchronization
Skeletal Muscle Reserve Cells: Isolation and Cell Cycle Analysis 167

4. Recombinant human basic Fibroblast growth Factor (bFGF)
(Promega): stock solution at 100 μg/ml in distilled water.
5. Nocodazole (Sigma, St Louis, MO, USA): stock solution at
10 mg/ml in DMSO (Sigma) (see Notes 1 and 2).
7. APC Brdu Flow kit (BD Biosciences).
8. Primary Human skeletal myogenic cells prepared as described
in Chazaud et al. [18].
9. Flow cytometer.
2.3 Materials 1. 100 mm petri dish (BD Biosciences).

for the Analysis 2. 50 ml conical centrifuge tubes (BD Biosciences).
of Human Myogenic
Cell Quiescence
5. 5 ml flow cytometry tubes (BD Biosciences).
6. Washing buffer: HBSS (1×) Mg2+/Ca2+ free supplemented
containing 2 % v/v heat-inactivated Fetal Bovine Serum (FBS)
(Gibco Life Technologies).
7. Pyronin Y (Sigma): stock solution at 10 mg/ml in distilled
water.
8. Hoechst 33342 (Sigma): stock solution at 10 mg/ml in
distilled water.
9. DNAse (BD Biosciences): stock solution at 300 μg/ml in
distilled water.
10. RNAse (Qiagen, Hilden, Germany): stock solution at
2 mg/ml in distilled water.
11. Primary Human skeletal myogenic cells prepared as described
in Chazaud et al. [18].
12. Flow cytometer.
2.4 Materials 1. 60 mm petri dish (BD Biosciences).

for Isolation 2. 50 ml conical centrifuge tubes (BD Biosciences, Franklin
of Murine RCs Lakes, NJ, USA).
3. 40 μm cell strainer (BD Biosciences).
4. Growth medium: DMEM high glucose, 4 mM L-glutamine,
sodium pyruvate, supplemented with 100 U/ml penicillin and
100 μg/ml streptomycin (Gibco Life Technologies) contain-
ing 15 % heat-inactivated Fetal Bovine Serum (Gibco Life
Technologies) and Chicken embryo extract (CEE) (Seralab,

Haywards Heath, UK).
5. Differentiation medium: DMEM high glucose, 4 mM
L-GLUTAMINE, sodium pyruvate, supplemented with 100 U/ml
penicillin and 100 μg/ml streptomycin (Gibco Life
Technologies) containing 4 % heat-inactivated Horse serum
(Gibco Life Technologies).
6. Matrigel (BD Biosciences) for coating culture dishes: Matrigel
is diluted to 1:5 in DMEM (see item 4), aliquoted and stored
at −20 °C.
9. Primary Murine skeletal myogenic cells prepared as described
in Collins and Zammit [19] or Pasut et al. [20].
3 Methods
All medium and reagents used should be pre-warmed before use

unless indicated.
3.1 Isolation 1. Plate primary human myogenic cells at a concentration of

of Human RCs 3,000 cells/cm2 in 100 mm plates in 10 ml of growth medium
(see Note 3).
2. Add bFGF at a final concentration of 10 ng/ml and grow cells
for 4 days at 37 °C. Replace growth medium every other day.
3. At 80 % of confluent density (see Note 4), remove growth
medium, wash twice with PBS (1×) and add 10 ml of differen-
tiation medium.
4. Incubate with differentiation medium for 10 days at 37 °C
(see Note 5). Change medium every other day.
5. Remove differentiation medium and wash cells three times
with PBS (1×).
6. Proceed for a short mild trypsinization by adding Trypsin-
EDTA 0.15 % (0.1 ml/cm2) (for 30 s to 1 min). Monitor myo-
tube detachment under microscope.
7. When all myotubes are detached, stop trypsin activity by add-
ing 5 ml of growth medium. Only quiescent mononucleated
undifferentiated RCs should remain attached to the culture
dish (see Note 6).
8. Transfer detached myotubes into a 50 ml conical centrifuge
tube containing 10 ml of growth medium, rinse with 5 ml of
growth medium and centrifuge cells for 10 min at 300 × g at
room temperature. Myotube preparation may be used for
molecular analyses (see Note 7).
9. Wash the culture dish containing attached RCs three times

with PBS (1×).
10. Trypsinate (0.1 ml/cm2 of Trypsin EDTA 0.25 %) for 3–5 min
at 37 °C. Check cell detachment under microscope.
11. Transfer detached cells into a 50 ml conical centrifuge tube
containing 10 ml of growth medium, rinse with 5 ml of growth
medium to inactivate trypsin activity.
12. Place a 40 μm cell strainer on top of 50 ml conical centrifuge
tube and transfer collected RCs through the filter. Wash filter
with 10 ml of growth medium (see Note 8).
13. Centrifuge cells for 10 min at 300 × g at room temperature.
14. Discard supernatant and resuspend cell pellet with 10 ml of
growth medium.
15. RCs may be used for molecular analysis or may be plated at a
concentration of 3,000 cells/cm2 in 100 mm petri dish in
10 ml of growth medium to give rise to new colonies (see
Notes 9 and 10).
3.2 Human Myogenic 1. Plate human primary skeletal myogenic cells at a concentration
Cell Synchronization of 3,000 cells/cm2 in 100 mm petri dish in 10 ml of growth
medium.
2. Add 100 μg/ml of bFGF at a final concentration of 10 ng/ml
and grow cells for 4 days at 37 °C.
3. Add Nocodazole to the culture at a final concentration of
1 μg/ml. Incubate for 16 h at 37 °C (see Notes 11 and 12).
4. Remove medium, wash cells twice with PBS (1×) and add
10 ml of fresh growth medium.
5. Assess cell synchronization by flow cytometry using BrdU
staining to monitor actively cycling cells, as described by APC
BrdU flow kit catalog provided by supplier. Briefly, myogenic
cells are incubated with BrdU at a final concentration of 10 μM
in growth medium for 16 h. Cells are subsequently fixed and
treated for staining with anti-BrdU as recommended by man-
ual supplier. BrDU stained cells are analyzed by flow cytometry
3.3 Analysis 1. Plate human primary skeletal myogenic cells at a concentration

of Human Myogenic of 3,000 cells/cm2 in 100 mm petri dish in 10 ml of growth
Cell Quiescence medium.
2. Add bFGF at a final concentration of 10 ng/ml and grow cells
for 4 days at 37 °C.
3. Remove growth medium and wash cells three times with
PBS (1×).
4. Trypsinize cells (0.1 ml/cm2 of Trypsin EDTA 0.25 %).
Fig. 1 Synchronization of human myogenic cells. G2/M cell cycle arrest following
Nocodazole treatment was confirmed using APC BrdU Flow kit, by measuring
cell-incorporated BrdU (anti-BrdU antibody) and total DNA content (7-AAD). Cells
incubated with only bFGF (a) present normal cell cycle profile with cells in G1/G0
(R4), S (R2 and R3), and G2/M (R5) phase (the small cell population in the bottom
left represent necrotic/apoptotic cells). Rare cells are observed in R3 region as
primary human myogenic cells exhibit a long cell cycle (more than the incubation
time with BrdU (16 h)). When cells are treated with Nocodazole (b), the majority
of myogenic cells are observed in G2/M phase (R5), confirming their synchroni-
zation and G2/M arrest after Nocodazole treatment

medium to inactivate trypsin and centrifuge cells for 10 min at
300 × g at room temperature.
6. Discard supernatant and resuspend cell pellet with 10 ml of
ice-cold washing buffer (HBSS, 2 % FBS).
7. Count cells and transfer 1×106 cells into 5 ml flow cytometry
tubes.
8. Centrifuge cells for 5 min at 300 × g at 4 °C.
9. Resuspend cells in 500 μl of Hoechst 33342 at a final concen-
tration of 10 μg/ml in HBSS/FBS for 30 min at 37 °C in the
dark (see Notes 14 and 15).
10. Add 500 μl of Pyronin Y at a final concentration of 2.5 μg/ml
in HBSS/FBS (see Notes 16 and 17).
11. Incubate with Pyronin Y solution for 30 min at 37 °C in the
dark (see Note 18).
12. Control cells were treated with RNAse at a final concentration
of 2 mg/ml in PBS (1×) for 2 h at 37 °C prior to Hoechst
staining. Other control cells were treated with DNAse at a final
concentration of 300 μg/ml in PBS (1×) for 1 h at 37 °C prior
to Pyronin Y staining.
13. Add 1 ml of ice-cold HBSS/FBS.
14. Centrifuge cells for 5 min at 300 × g at 4 °C.
15. Resuspend cells in 1 ml of ice-cold HBSS/FBS, keep cells on
ice in the dark and proceed to flow cytometer analysis (see
Notes 19 and 20) (Fig. 2).
3.4 Isolation Obtaining Satellite cells. We use two different approaches, previ-
of Murine RCs ously described, to isolate pure satellite cells:
(A) Cultures of isolated myofibers prepared from EDL and Soleus
muscles [19]. Single myofibers are separated from intact mus-
cles following collagenase digestion and mechanical triturat-
ing. Single myofibers are plated on Matrigel, and proliferating
satellite cell progenies migrate out from the host myofiber after
2 days of plating.
(B) Cultures of FACS-sorted satellite cells prepared from limb
muscles [20]. Satellite cells are isolated from hindlimb muscles
following collagenase–dispase digestion. Then, satellite cells
can be isolated based on negative selection for CD45, CD31,
and Sca1, and positive selection for CD34 and α7-integrin.
1. Coat petri dishes with Matrigel. Thaw an aliquot by placing it
on ice for at least 30 min to allow the Matrigel to completely
liquefy. Use a chilled glass pipette to draw up the diluted Matrigel
solution and coat the dishes (1 ml per 60 mm petri dish).
Fig. 2 Evaluation of the quiescence of human myogenic cells with Hoechst and
Pyronin Y. Non-treated human myogenic cells show cells present in all phases of
the cell cycle (b) while the pyronin Y labelling is lost after RNAse treatment (a).
When Ang1, a promotor of RC quiescence, is added, the number of cells in G0 is
increased (c) while when bFGF, a promotor of cell cycle, is added, the number of
cells in G1 is increased (d)
Let the solution in the dish, on ice, for 2–3 min, and then use
the same chilled pipette as before to remove the Matrigel solu-
tion and place it back in the aliquot tube that was kept on ice.
This will create a thin Matrigel coating at the bottom of the
dishes.
2. Plate cells in the Matrigel-coated culture dishes in 2 ml of
growth medium. When using 35-mm dishes: if starting from
FACS-sorted cells, plate 2 × 104 cells, if starting from isolated
myofibers, plate 50 fibers.
3. Grow cells for 9 days at 37 °C and replace growth medium
every 3 days (see Notes 21–23).
4. At day 9, cells should be at 80 % of confluent density (see Note
24), remove growth medium, wash twice with PBS (1×), and
add 2 ml of differentiation medium.
5. Incubate with differentiation medium for 6 days at 37 °C
(see Note 25).
6. Remove differentiation medium and wash cells three times
with PBS (1×).
7. Trypsinate (0.1 ml/cm2 of Trypsin EDTA 0.25 %) for 3–5 min
at 37 °C. Monitor cell detachment under microscope
(see Note 26).
medium to inactivate trypsin activity.
9. Place a 40 μm cell strainer on top of 50 ml conical centrifuge
tube and transfer collected cells through the filter. Wash filter
with 10 ml of growth medium.
10. Centrifuge cells that have passed through the filter (RCs) for
10 min at 300 × g at room temperature.
11. Discard supernatant and resuspend RC cell pellet with 10 ml
of growth medium.
12. RCs may be used for molecular analysis or may be plated to
give rise to new colonies.
13. Myotubes collected in the top of the filter may be recovered for
molecular analyses. Place the filter containing the myotubes in a
Petri dish, add some medium and collect the cells in the filter.
4 Notes
1. Suspend Nocodazole powder in DMSO at a concentration of

10 mg/ml. Warm solution to dissolve. Keep stocks at −20 °C
and avoid freeze–thaw cycles.
2. Nocodazole is mutagen, to be handled carefully and with

gloves.
3. Primary human myogenic cells should be passed maximum for
3 times as they start to lose their myogenic potential.
4. bFGF enhances myogenic cell growth and proliferation. When
added to growth medium, myogenic cell subconfluency is
reached within 3–4 days otherwise cell subconfluency is
reached within 7 days approximatively [21].
5. After 10 days of differentiation, about 80–85 % of myogenic
cells fuse to form multinucleated differentiated myotubes while
~15–20 % of myogenic cells do not differentiate, remain mono-
nucleated, stop proliferating, and return to quiescence. The
latter are named Reserve cells (RCs). They are located in adja-
cent position to myotubes [12].
6. Mild and short trypsination is used because myotubes are more
sensitive to trypsinization than RCs [10]. Incubation should
be short as longer incubation with Trypsin-EDTA 0.15 % may
cause detachment of mononucleated cells, RCs.
7. Myotube preparation may be replated but with various adhe-
sion efficiency. Myotubes do not grow as myotube myonuclei
are postmitotic. For immunocytochemistry purposes, it is pref-
erable to use fully differentiated cultures containing both RCs
and myotubes that can be easily distinguished by differential
marker staining.
8. Filtering ensures no myotube contamination of RC prepara-
tion. It is possible to check if myotubes are retained on the
40 μm filter after step 13 by taking an aliquot and check under
the microscope.
9. RCs can be replated. Once plated in growth medium, RCs will
activate and spontaneously proliferate to form myoblasts. Under
differentiation conditions, myoblasts differentiate and fuse to
give rise to both differentiated myotubes and new quiescent RCs.
10. RC identity is confirmed by Pax7 expression level, Pax7 identi-
fied as a marker of quiescent, activated, and proliferating myo-
genic cells [22]. See Fig. 3.
11. Control cells were treated with DMSO at the same concentra-
tion used for Nocodazole to demonstrate that DMSO do not
alter cell-cycle phase distribution neither cell proliferation.
12. Longer incubations with Nocodazole and higher concentrations
of Nocodazole increase toxicity and irreversible arrest [24].
13. Nocodazole-arrested cells exhibit larger size than non-arrested
cells (our observations and ref. [23]).
14. Cells are permeabilized by heat shock (from cold to warm
37 °C) to ensure proper incubation with Hoeschst 33342 and
Pyronin Y working solutions.
Fig. 3 Pax7 expression by human myogenic cells. RT-qPCR analysis of Pax7 in

isolated RCs, myotubes and whole culture of proliferating myogenic cells. Pax7
is highly expressed by quiescent mononucleated Reserve cells (RC) and prolifer-
ating myoblasts (Mb) compared to multinucleated differentiated myotubes (MT)
15. To avoid cell clumping, we add EDTA at a final concentration of

0.5 mM to Hoeschst 33342 and Pyronin Y working solutions.
16. Pyronin Y also stains DNA. The initial step of Hoechst staining pre-
vents DNA staining by Pyronin Y. Pyronin Y is used to stain RNA
after the binding of DNA is blocked with Hoechst 33342 [16].
17. We use 2.5 μg/ml as optimal concentration of Pyronin Y for
RNA staining. Higher concentrations of Pyronin Y may induce
RNA denaturation and condensation.
18. We find 30 min of incubation with Pyronin Y is optimal to stain
RNA in primary human myogenic cells, longer incubation with
Pyronin Y will induce Pyronin Y intercalation to DNA.
19. Hoeschst 33342 fluoresces in blue. Hoechst 33342 can be
excited at 350 nm. Maximal emission of Hoechst 33342 fluo-
rescence is at 461 nm.
20. Pyronin Y fluoresces in orange-red. Pyronin Y can be excited
between 488 and 530 nm. Maximal emission of Pyronin Y
fluorescence is at 570 nm.
21. After 3 days of culture, colonies of about 20 cells should have
grown.
22. After 6 days of culture, all proliferating cells are myoblasts that
express desmin (muscle cytoskeleton marker).
23. If myogenic cells are isolated from plated myofibers, myofibers
may have shrunk after 3–5 days in culture.
24. After 9 days of culture in growing medium, about 60 % of
myogenic cells have fused and formed multinucleated myotubes.
Proliferating mononucleated cells can be visualized between
Fig. 4 Culture of murine cells showing differentiated myotubes and side reserve
cells. After differentiation step, murine cell cultures are labelled for the transcrip-
tion factor Pax7 that marks quiescent and proliferating myogenic cells and for
Myosin Heavy Chain (MyHC) that is expressed by fullly differentiated myogenic
cells. Pax7pos MHCneg cells are located beside Pax7neg MHCpos large myotubes
the myotubes, and express the myogenic transcription factors

Pax7 and/or MyoD proteins [22].
25. After 3 days in differentiation medium, Pax7+/MyoD- reserve
cells are located around the multinucleated myotubes (see Fig. 4).
26. Mild trypsination is not useful with murine cells as some RCs
will remain stuck on the dish while other detach with myo-
tubes. Thus, mild trypsination would necessitate also a filtra-
tion step to separate myotubes from RCs.
Acknowledgments
We thank Jyotsna Dhawan for her advices concerning the synchro-

nization of myogenic cell cultures. We thank Marie-Claude
Gendron for the setting up of the flow cytometry analysis of
Pyronin/Hoechst staining.
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Chapter 15
Modulation of the Host Skeletal Muscle Niche

for Donor Satellite Cell Grafting
Luisa Boldrin and Jennifer E. Morgan
Abstract
Skeletal muscle tissue has a remarkable capability of regenerating in pathological conditions or after injury.
The principal muscle stem cells, satellite cells, are responsible for this prompt and efficient process.
Normally quiescent in their niches underneath the basal lamina of each muscle fiber, satellite cells become
activated to repair or form new fibers. Ideally, healthy donor stem cells could be transplanted to regenerate
the skeletal muscle tissue to repair a genetic defect. However, to be efficient, cell grafting requires modula-
tion of the host muscle environment to allow homing of, and regeneration by, donor satellite cells. Here,
we provide methods to modulate the host mouse muscle environment in order to destroy or preserve the
muscle niche before transplanting donor satellite cells. We also describe methods to investigate donor-
derived muscle regeneration and self-renewal.
Key words Stem cells, Satellite cells, Skeletal muscle, Muscle regeneration, Differentiation,
Self-renewal
1 Introduction
Under normal physiological conditions, adult skeletal muscle is a

relatively stable tissue, consisting of long, cylindrical, multinucleated
muscle fibers (myofibers), which contain the contractile elements.
Muscle fiber nuclei (myonuclei) are peripherally located under the
sarcolemma of the myofiber and are postmitotic (Fig. 1) [1].
Satellite cells are located in niches between the basal lamina and the
sarcolemma of each fiber. These are normally quiescent stem cells
[2] and can be identified by the expression of specific satellite cell
markers, with the majority of quiescent satellite cells expressing
Pax7 [3]. Following injury, muscle fibers are destroyed and a
regenerative process occurs. In the mouse muscle after injury
induced by, for example, the injection of a myotoxin [4, 5], myofibers
undergo necrosis, inflammatory cells enter the muscle and satellite
cells are activated. Three days after injury, necrotic muscle fibers are
replaced by mononucleated cells, some of which are inflammatory
179
180 Luisa Boldrin and Jennifer E. Morgan
Fig. 1 Histology of transverse sections of murine wild type muscle, stained with Hematoxylin and Eosin. In
uninjured muscle, myofibers are mature, with nuclei in a peripheral position. Three days after myotoxin
(notexin) injection, muscle fibers are destroyed. Four days after injury, myotubes begin to form. Five days after
injury, newly formed, small, basophilic, centrally nucleated fibers are present. Scale bar = 100 μm
cells and others the progeny of satellite cells (myoblasts). Myoblasts

proliferate and eventually either fuse together to form new myofi-
bers, or fuse with damaged fibers to repair areas of focal necrosis.
This process occurs rapidly and efficiently: a few days after damage,
newly regenerated myofibers, distinguished by their small size,
basophilic cytoplasm and myonuclei in a central position, are
detectable (Fig. 1). These regenerated fibers mature and increase
in size; some myonuclei migrate to a peripheral position, but, in
the mouse, the majority of nuclei in regenerated fibers remains
centrally located, an useful indicator that the fiber has undergone
regeneration at some time in the past [6].
In muscular dystrophies, such as the most common Duchenne
Muscular Dystrophy (DMD) in which myofibers are fragile due to
the lack of functional dystrophin protein, muscle regeneration
occurs, but the congenital defect leads to continuous cycles of
degeneration and regeneration. Eventually, regeneration fails and
muscle is substituted by fibrous/adipose/connective tissue [7].
The mdx mouse models this pathology of muscle degeneration and
regeneration, allowing investigation of approaches aimed to treat
the genetic deficit. Ideally, replacement of dystrophic satellite cells
with healthy donor satellite cells would generate dystrophin posi-
tive myofibers. However, integration of donor cells in the host tis-
sue requires modulation of the muscle environment. In particular,
we have found that transplantation of donor satellite cells in mdx
muscles is successful only if the niche is preserved and host satellite
cells are incapacitated [4]. We have tested donor-derived muscle
regeneration in host muscles where the endogenous satellite cell
niche was either destroyed (by injection of myotoxins—i.e.
cardiotoxin, notexin, barium chloride—or by cryoinjury) or pre-
served (by irradiation). Here, we describe these methods of muscle
niche modulation, of isolation and grafting of a pure population of
satellite cells and investigation of donor-derived muscle formation
after transplantation of donor satellite cells.
Modulation of Muscle Environment 181
2 Materials
2.1 Host and Donor Breeding of mice and experimental procedures were carried out in
Mouse Strains the Biological Services Unit of University College London,
Institute of Child Health, in accordance with the Animals (Scientific
Procedures) Act 1986. Experiments were carried out under Home
Office licence.
2.1.1 Host Mouse Strain The mdx mouse, which is the genetic and biochemical homologue
of human Duchenne Muscular Dystrophy [8], was crossed to a
nude (Foxn1–/–) background to generate dystrophin-deficient,
immunodeficient mdx nu/nu mice [9]. This is a valuable mouse
model for cell transplantation, as the lack of T cells prevents immu-
nological rejection of donor cells. Furthermore, in mdx nu/nu
mice aging occurs prematurely, making them a good model to
study an advanced stage of dystrophy [10]. In transplantation
experiments, as extensive degeneration and regeneration occurs in
mdx at 3 weeks of age, this is an optimal time to graft donor cells.
2.1.2 Donor Mouse To investigate the contribution of donor cells to muscle regenera-
Strains tion, we isolate donor cells from the 3F-nlacZ-2E transgenic
mouse, where all the myonuclei that express myosin light chain-3F
also express β-Gal [11] (Fig. 2). Satellite cells in these mice are
β-Gal negative [10, 11] (Fig. 2).
To investigate if grafted donor cells give rise to satellite cells,
we use the Myf5nlacZ/+ mouse to isolate donor cells. In this mouse,
the majority of satellite cells expresses, β-Gal [12, 13]. β-Gal
expression in nuclei underneath the myofiber basal lamina marks
donor-derived satellite cells (Fig. 2). However, it should be taken
into account when performing analyses that newly regenerated
myonuclei retain Myf5nlacZ expression for a short time [14, 15].
To investigate integration of donor cells in muscle, inside and/
or outside the myofibers, we use the β-actin-Cre:R26NZG mouse
(obtained from crossing a homozygote male β-actin-Cre (FVB/
N-Tg(ACTB-cre)2Mrt/J)—a kind gift from Massimo Signore,
UCL—with an homozygote female R26NZG (Gt(ROSA)26Sortm1(CAG-
lacZ,-EGFP)Glh
) (The Jackson Laboratory, USA)). Nuclei underneath
the basal lamina of myofibers expressing β-Gal can be either myo-
nuclei or satellite cells. If found outside the myofiber basal lamina,
they can be myoblasts or other cell types (Fig. 2).
2.2 Pre-treatment 1. Injectable anesthetic: solution of Hypnorm (79 μl/ml fentanyl

of Host Skeletal citrate, 2.5 mg/ml fluanisone, Janssen-Cilag Ltd) and
Muscle Tissue Midazolam (1.25 mg/ml midazolam, CP Pharmaceuticals
Ltd) diluted 1:4 in water.
2.2.1 Irradiation
Fig. 2 Single fibers isolated from different donor mouse models. In the 3F-nlacZ-2E myofiber, only the myonu-
clei are X-gal stained. The X-gal negative, DAPI positive nucleus on the fiber (arrow) is a satellite cell. In the
Myf5nlacZ/+ myofiber, only satellite cells are X-gal positive; note that a strong X-gal staining can quench DAPI
signal (arrow). In the β-actin-Cre:R26NZG myofiber, all the nuclei are X-gal positive. Size bar = 10 μm
2. Custom made lead blocks, to shield—by 4 cm of lead—the

body of the mouse so that only the hind limbs are exposed to
radiation.
3. 30 g needle mounted on 500 μl syringes for subcutaneous
injections of anesthetic.
2.2.2 Myotoxin Injection Administration of substances below reported was performed

according to LASA good practice guidelines (www.lasa.co.uk).
1. Inhalable anesthetic (isoflurane) and apparatus to anesthetize
mice, or injectable anesthetic (as detailed in Subheading 2.2.1).
2. Notexin (Notechis scutatus notexin, 10 μg/ml, Latoxan): 10 μl
per tibialis anterior (TA) muscle.
3. Cardiotoxin (10 μM, Latoxan): 50 μl per TA muscle.
4. Barium Chloride (BaCl2) (1.2 % solution in PBS): 25 μl per TA
muscle.
5. Vetergesic: administer at a concentration of 0.05 mg/kg. If a

volatile anesthetic is used, administer it before, or at time of,
anesthesia. If hypnorm/hypnovel anesthesia is used, give
Vetergesic at the end of a surgical procedure, as it should
reverse the effect of fentanyl.
6. 29 g needles mounted on 500 μl syringes for intramuscular
myotoxin injection.
7. 30 g needles mounted on 500 μl syringes for subcutaneous
injection of Vetergesic.
2.2.3 Cryoinjury 1. Inhalable anesthetic (isoflurane) and apparatus to anesthetize

mice.
2. Sterile scissors.
3. Copper cryoprobe chilled in liquid nitrogen.
4. VICRYL rapide absorbable suture (6/0, W9913, Johnson &
Johnson Medical Ltd).
5. Vetergesic (as detailed in Subheading 2.2.2).
2.3 Donor Satellite 1. Preparation of single donor myofibers requires materials

Cell Preparation detailed in [16].
2. Plating medium: DMEM/ L-Glutamine (final concentration
4 mM)/Penicillin-Streptomycin (final concentration: penicil-
lin: 100 units/ml; streptomycin: 100 μg/ml) supplemented
with 10 % horse serum (Invitrogen) and 0.5 % chicken extract
embryo (Sera Laboratories).
3. 19 g needle mounted on 1 ml syringe for isolation of satellite
cells from myofibers (stripping).
4. 40 μm cell sieves to separate myofibers from stripped satellite
cells.
5. Sterile Falcon tubes (BD Biosciences) to centrifuge collected
cells.
6. Refrigerated centrifuge.
2.4 Donor Satellite 1. No. 11 scalpel.

Cell Grafting 2. Microcapillary pipettes (Drummond Microcaps), pulled by
using flame and forceps and oven-sterilized.
2.5 Test of 1. Inhalable or injectable anesthetic.

Functionality 2. Notexin.
of Donor-Derived
3. Vetergesic.
Satellite Cells
4. Needles and syringes (all detailed in Subheading 2.2.2).
2.6 Detection of 1. Gum Tragacanth (Sigma G-1128) and cork disks to mount
Regenerated Muscle harvested muscles (as detailed in ref. [16]).
Fibers of Donor Origin 2. Isopentane and liquid nitrogen.
2.6.1 Muscle Freezing
2.6.2 Analysis of 1. Glutaraldehyde: 0.5 % (v/v) solution in PBS.

Donor-Derived Fibers 2. Washing solution: 2 mM MgCl2 in PBS.
3. Detergent: PBS containing 0.01 % (v/v) Sodium Deoxycholate,
0.02 % (v/v) Nonidet P40 (IGEPAL CA-630), 2 mM MgCl2.
4. X-gal stock: 40 mg/ml X-gal (5-Bromo-4-chloro-3-indolyl
β-D-galactopyranoside) in dimethyl sulfoxide.
5. X-gal diluent: PBS containing 0.01 % (v/v) Sodium
Deoxycholate, 0.02 % (v/v) Nonidet P40, 2 mM MgCl2,
5 mM K3Fe(CN)6 and 5 mM K4Fe(CN)6.
6. Polysine-coated glass slides.
7. Materials for immunostaining: antibody against dystrophin
protein and appropriate secondary antibody, 10 % (v/v) serum
in PBS (the serum should be from the same species in which
the secondary antibody was raised) for blocking, fluorescent
mounting medium (DAKO) and 4′,6-diamidino-2-
phenylindole (DAPI) fluorescent dye (final concentration
0.1 μg/ml).
2.6.3 Analysis of 1. 4 % paraformaldehyde (PFA) in PBS.

Donor-Derived Satellite 2. 2 mM MgCl2 in PBS.
Cells
3. X-gal stock (as detailed in Subheading 2.6.2).
4. X-gal diluent (as detailed in Subheading 2.6.2).
3 Methods
3.1 Pre-modulation 1. Anesthetize mice with a subcutaneous injection of 50 μl

of Recipient Mouse Hypnorm and Midazolam solution.
Muscles 2. Place fully anesthetized mice on the lead block, so that the
3.1.1 Irradiation body and the tail are shielded by 4 cm of lead and the hind
limbs are on a plastic platform. Tape feet securely to the plat-
form, to hold legs in place. Cover the mouse body with cotton
wool to keep them warm during the procedure.
3. Irradiate the legs with 18 Gy at a rate of 0.72 Gy/min [17].
A different dose of radiation may also promote donor-derived
regeneration, provided it incapacitates endogenous satellite
cells maintaining the integrity of the niche (see Note 1) [4].
4. Remove mice from the irradiator as soon as irradiation is com-
pleted and keep warm until they have recovered from the anes-
thetic. Place mice back in the cage with dampened food.
3.1.2 Surgical 1. Surgical procedures described below require mouse anesthesia

Procedures with inhalable or injectable anesthesia (see Note 2).
2. Tape down mouse leg, with TA uppermost and sterilize leg
with 70 % alcohol.
3. Analgesia must be administered—timing according to the
anesthetic used (see Subheading 2.2.2).
4. Perform surgical procedure as described below.
5. Keep mice warm and supervised until they have recovered
from the anesthetic.
6. Putting dampened food and sterile water facilitates feeding
after muscle injury.
7. If mice are not placing their feet correctly, or show persistence
of adverse effects by day 4 after myotoxin injection, they should
be culled by a schedule 1 procedure.
Myotoxin Injury 1. Inject the myotoxin percutaneously into the TA of the anes-
thetized mouse in the volume and concentration described in
Subheading 2.2.2.
Cryoinjury 1. Make a skin incision in the leg of the anesthetized mouse to

expose the muscle to be injured.
2. Freeze the muscle to be injured with a copper cryoprobe
chilled in liquid nitrogen.
3. Hold the cryoprobe against the top of the TA muscle for 10 s,
until the area is frozen. Then remove it, allow the muscle to
thaw completely and freeze the bottom half of the TA for 10
seconds. Repeat the freeze-thaw procedure twice. The muscle
should be thawed completely before cells are injected.
4. Close the skin incision by suturing.
3.2 Preparation of a 1. After pooling all fibers in a single dish with plating medium,
Pure Population of release satellite cells by physical trituration for 5 min with a
Donor Mouse Satellite 19G needle mounted on a 1 ml syringe. Pass cell suspension
Cells through 40 μm cell filter to remove hypercontracted fibers.
Counting of stripped satellite cells is very difficult and ambigu-
ous due to the large amount of debris sized similarly as the very
small freshly-isolated satellite cells. As the number of satellite
cells per fiber is known [15], counting the number of fibers
before stripping allows the expected number of satellite cells to
be estimated [4, 10, 18].
2. If it is necessary to reduce the volume of medium containing
stripped satellite cells, perform two rounds of centrifugation:
the first at 240 × g for 15 min, to collect bigger cells, the sec-
ond at 600 × g for 20 min at 4 °C to collect the smaller cells (see
Note 3). Collect both pellets and mix them in the desired
volume of medium.
3.3 Grafting of 1. Anesthetize mice with isoflurane.

Donor-Satellite Cells 2. Inject muscles that had been pre-treated with irradiation or
in Pre-treated Host myotoxin injection or cryoinjury either on the same day, or up
Muscles to 3 days before grafting.
3.3.1 Cell Grafting 3. Place mouse on a sterile drape over a hot pad and tape down
leg so that the TA is uppermost.
4. Wipe leg with 70 % alcohol to sterilize and with a no. 11 scal-
pel make a small incision (<1 mm) in the skin above the TA
muscle to facilitate the insertion of the capillary into the TA
muscle.
5. Using a stereomicroscope, inject the cells in a volume 3–5 μl as
a larger volume can leak out after injection (see Note 4).
6. Repeat the procedure with the contralateral leg if required
(see Note 5).
7. Once injection(s) have been performed, keep the mouse warm
until it has recovered from the anesthetic and replace into the
cage.
3.3.2 Test of Donor- 1. Three weeks after cell grafting (see Note 6), anesthetize mice
Derived Satellite Cell and inject notexin into TA muscle(s) as described in
Functionality Subheadings 3.1.2 and 3.1.2.1.
3.3.3 Analysis of Grafted 1. Donor-derived muscle formation is typically analyzed 4 weeks

Host Muscles after cell grafting, as this allows sufficient time for muscle
regeneration [15]. As dystrophin-positive fibers within mdx
Detection of
muscles might be revertant fibers of host origin [19], rather
Donor-Derived Nuclei
than muscle fibers of donor origin, a second marker of muscle
of donor origin is required [4, 10, 15, 18]. An area of dystro-
phin positive fibers is taken to be of donor origin if donor
myonuclei (visualized in a serial section) are present in that
area of muscle (Fig. 3). When the regenerated grafted muscle
is destroyed by notexin 3 weeks after cell grafting, finding of
newly regenerated fibers (expressing neonatal myosin) [20] of
donor origin (expressing dystrophin or containing nuclei of
donor origin) 1 week later is evidence that the donor cells had
given rise to functional muscle stem cells, that had contrib-
uted to muscle regeneration after notexin injury (Fig. 4)
[4, 10, 15, 20].
2. Mice are killed by a schedule 1 procedure and TA muscles
removed, bisected transversely and mounted on cork disks in
Gum Tragacanth (as detailed in ref. [16]).
3. Freeze muscles in isopentane cooled in liquid nitrogen. Once
freezing is completed, transfer the samples to a −80 °C freezer.
4. Using a cryostat, cut transverse 7 μm serial cryosections at
100 μm intervals through the entire muscle and collect sections
Fig. 3 Detection of donor-derived muscle regeneration. (a) When 3F-nlacZ-2E satellite cells are grafted in mdx
nu/nu mice, their contribution to muscle regeneration is demonstrated by the presence of X-gal positive myo-
nuclei inside fibers that express dystrophin (shown by immunostaining in a serial section). (b) Grafting of
β-actin-Cre:R26NZG satellite cells allows us to determine whether satellite cells give rise to cells other than
satellite cells or myofibers. (c) Grafting of satellite cells isolated from Myf5nlacZ/+ myofiber allows the detection
of donor-derived satellite cells on myofibers (5× magnification). Identification of donor-derived satellite cells
can be performed on transverse sections: these are cells that are X-gal positive and are at the myofiber periph-
ery (arrows), underneath the basal lamina (stained by laminin antibody). Nuclei counterstained by DAPI. Size
bar = 50 μm
on polysine-coated glass slides. Air dry before storing in a

−80 °C freezer.
5. To detect donor-derived nuclei, fix muscle sections in 0.5 %
glutaraldehyde for 10 min on ice, rinse in cold 2 mM PBS–
MgCl2 and, after a quick wash in 2 mM PBS–MgCl2, incubate
Fig. 4 Test of functionality of donor-derived satellite cells. In a grafted mdx nu/nu muscle that has been left for
3–4 weeks to allow complete regeneration, myofibers of both host and donor origin are destroyed by notexin
injection. This promotes satellite cell-mediated regeneration. (a) Schematic representation of muscle sections
1 week after notexin injection: fibers that have not been destroyed by notexin are of larger diameter with nuclei
in a peripheral position (in grey ) and may be of either host (dystrophin negative) or donor (dystrophin positive
in red ) origin. Fibers that are regenerating 7 days after notexin injury are small, centrally-nucleated and
strongly express neonatal myosin (green ); these may be either of host (dystrophin negative) or donor (dystro-
phin positive in red ) origin. (b) Newly regenerated donor-derived myofibers in a grafted and notexin-injured
host muscle immunostained with neonatal myosin (green ) and dystrophin (red ) antibodies. Nuclei counter-
stained by DAPI. Size bar = 50 μm
on ice for 10 min first in 2 mM PBS–MgCl2 then in cold deter-

gent and finally incubate in X-Gal solution overnight at 37 °C.
6. Wash in PBS and distilled water and, after mounting slides
with aqueous mounting medium and coverslips, observe them
under a microscope in bright field.
7. If X-gal positive nuclei are present, proceed with dystrophin
immunostaining on serial sections. When muscles have been
notexin-injured after grafting, a neonatal myosin antibody
should also be used in co-staining with dystrophin antibody.
After blocking for 30 min, muscle sections are incubated for an
hour at room temperature with primary antibodies, followed,
after three 10 min washes in PBS, by an hour-incubation with
appropriate secondary antibodies at room temperature. Mount
slides with fluorescent mounting medium and DAPI before
analyses under the microscope.
Detection of Donor-Derived 1. For detection of donor-derived satellite cells on longitudinal

Satellite Cells myofibers: fix the whole muscle in 4 % PFA for 15 min, incu-
bate in PBS for 30 min and then in 2 mM PBS–MgCl2 for
20 min (see Note 7). Finally, incubate in X-gal solution at
37 °C overnight. Rinse three times with PBS. β-Gal positive
satellite cells can be detected on myofibers by using a stereomi-
croscope (Fig. 3).
2. For detection of donor-derived satellite cells on transverse sec-
tions: whole X-gal stained muscles can be cryopreserved over-
night in 30 % (v/v) sucrose-PBS solution at 4 °C.
3. Proceed as described in Subheading 3.3.3, step 1 for muscle

freezing and cryosectioning.
4. Choose sections with the highest number of X-gal positive
Myf5nlacZ/+ nuclei and immunostain sections serial to these with
laminin antibody, as described in Subheading 3.3.3, step 2.
β-gal positive nuclei underneath the basal lamina of dystrophin
positive fibers, or of fibers in close proximity to dystrophin
positive fibers (see Note 8), are satellite cells of donor origin.
Blue nuclei in different positions can be myoblasts (if outside
the fibers), or nuclei of newly generated fibers (if in a central
position inside the fibers).
4 Notes
1. Time and dose of irradiation are critical variables for cell graft-
ing [4, 17]. A radiation dose higher than 18 Gy can also pro-
mote donor cell engraftment if host satellite cells are
incapacitated but not totally depleted, thus preserving a func-
tional host niche. Three days after 25 Gy irradiation of host
muscle, nearly all host satellite cells are depleted, whilst on the
day of radiation at least some host satellite cells remain in their
niches. In keeping with the necessity of preserving the host
muscle satellite cell niche, 25 Gy radiation promotes donor-
derived muscle regeneration only if applied on the day of,
rather than 3 days before, grafting. Similarly, using 18 Gy irra-
diation, a comparable amount of donor-derived muscle is
formed if irradiation is applied up to 3 days but not 4 weeks
before cell grafting, when nearly all host satellite cells are
depleted [4].
2. As recovery is always quicker in the case of inhalable anesthe-
sia, this is to be preferred to injectable anesthesia whenever
possible.
3. It is preferable to use a refrigerated centrifuge at 4 °C. This
helps to maintain cell viability.
4. Keep cells on ice after isolation until injection.
5. In an in vivo experiment, the best control is the muscle of the
contralateral leg (either untreated or treated with medium alone).
6. It is necessary to wait a sufficient time for muscle regeneration
to have occurred, before analysis or performing a further
procedure—usually 3–4 weeks.
7. Fixation can be compromised if pH of PFA is not adjusted
to 7.5.
8. Dytrophin production in host fibers regenerated by donor cells
is segmental: only fragments of fibers where donor nuclei are
integrated express dystrophin protein [14].
Acknowledgments
The authors thank Miss Rowan Asfahani for the pictures presented
in Fig. 1. This work was supported by Muscular Dystrophy
Campaign (grant code RA3/776) and Wellcome Trust University
Award (grant code 08241/Z/07/Z).
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Chapter 16
Isolation of c-Kit+ Human Amniotic Fluid Stem

Cells from Second Trimester
Michela Pozzobon, Martina Piccoli, Andrea Alex Schiavo,
Anthony Atala, and Paolo De Coppi
Abstract
Amniotic fluid-derived stem (AFS) cells have been described as an appealing source of stem cells because
of their (1) fetal, non-embryonic origin, (2) easy access during pregnancy overcoming the ethical issues
related both to the use of human embryonic cells and to the postnatal tissue biopsy with donor site mor-
bidity, and (3) their undemanding ability to be expanded. We and others have demonstrated the broad
differentiation potential and here we describe the established protocol we developed to obtain c-Kit+
human AFS cells, starting from second trimester amniocentesis samples.
Key words Human stem cells, Amniotic fluid stem cells, Magnetic selection, c-Kit, Fetal cells, CD117
1 Introduction
Amniotic fluid samples are commonly obtained following proce-

dures that pregnant women undergo around the second trimester of
gestation, namely, amniocentesis and amnioreduction. During the
first procedure, few milliliters (2–3 mL) are available for research.
Differently during amnioreduction, which is usually performed dur-
ing the third trimester in case of polydramnios, more than 500 mL
can be retrieved for stem cells extraction. Herein, we have focused
on obtaining a standard method to isolate AFS cells from amniocen-
tesis considering their continuous availability and easiness of collec-
tion. We select cells from fresh samples obtained just after the
procedure or from adherent cells after cytogenetic analysis.
AFS cells are selected for the expression of CD117 (Mast/stem
cell growth factor receptor also known as c-Kit) cells and share
characteristics of both embryonic and adult stem cells [1–3], and
they are able to differentiate toward different lineages such as
191
192 Michela Pozzobon et al.
CD117+ selection
Centrifuge and Resuspend and

remove supernatant seed on petri dish
Flow cytometry Expansion

analysis
Fig. 1 Schematic representation of the procedure to obtain and expand CD117 positive cells from fresh amni-
otic fluid samples. After the first centrifugation you can use a fraction of the cell pellet to analyze by flow
cytometry the level of markers present in the total population before culture and selection
adipose, bone, nerve [4], muscular [5, 6], vascular, and hematopoietic
tissues [7]. This selection allows the purification of a more homog-
enous population of cells from the amniotic fluid, which is usually
populated by several cell types [8]. Considering the low number of
CD117 positive cells in the amniotic fluid (range 1–5 %) we devel-
oped an accurate step-by-step methodology for their selection
which take in account both their low number and the limited
amount of fluid obtained at amniocentesis (see Fig. 1).
2 Materials
2.1 Culture Medium 1. Prepare the pre-selection medium: using a sterile syringe
and Plastic Ware plugged to a needle, inject 10 mL of sterile distilled water to
the Chang Medium C Lyophilized Supplement (catalog num-
ber T101-019) (IrvineScientific, Santa Ana, CA, USA) and let
equilibrate until no powder is visible inside the vial.
2. Transfer reconstituted Chang Medium C Supplement in the
bottle of Chang Medium B Basal (catalog number T101-019)
(IrvineScientific, Santa Ana, CA, USA) making a 100 mL final
volume of medium called “Chang B + C.” This medium should
be completed with your antibiotics (Penicillin and Streptomycin,
see Note 1) and also the L-Glutamine (2 mM final) (catalog
number 25030) (Gibco-BRL, Bethesda, MD, USA) at the
moment of use.
3. Expansion medium: MEM Alpha (catalog number A10490-01)
(Gibco-BRL, Bethesda, MD, USA) containing 20 % Chang
B + C, 15 % Fetal Bovine Serum (FBS, catalog number 10106-
169) (Gibco-BRL, Bethesda, MD, USA), antibiotics, and
L-Glutamine (2 mM final) (see Note 2).
Isolation of c-Kit+ Human Amniotic Fluid Stem Cells from Second Trimester 193
4. Trypsin 0.05 % with EDTA and Phenol Red (catalog number

25300) (Gibco-BRL, Bethesda, MD, USA).
5. 24-Well multiwell plates, non-treated, polystyrene, flat bottom
(catalog number 351147) (BD Falcon, Franklin Lakes, NJ,
USA). When growing these cells in culture it is very important
not to use “tissue culture treated” plates (multiwell or petri
dish) use only non-treated plates.
6. 35 mm × 10 mm dishes (catalog number 351008) (BD Falcon,
Franklin Lakes, NJ, USA).
7. 100 mm × 20 mm dishes (catalog number 351029) (BD
Falcon, Franklin Lakes, NJ, USA).
8. 150 mm × 25 mm dishes (catalog number 351013) (BD
Falcon, Franklin Lakes, NJ, USA).
9. 15 mL polypropylene conical centrifuge tube (catalog number
352096) (BD Falcon, Franklin Lakes, NJ, USA).
10. Round glass coverslips (35 mm of diameter).
2.2 Immune- 1. We recommend to select CD117 positive cells by an immuno-

Selection Components magnetic separation method, following manufacturer’s instruc-
tion and adapting it for slow number of cells (see Note 3): here
we describe the procedure using the CD117 MicroBead Kit
(catalog number 130-091-332) and the other required instru-
ments from Miltenyi Biotec Inc., Auburn, CA, USA.
2. MS MACS® Columns (catalog number 130-042-201).
3. MiniMACS™ Separator (catalog number 130-042-102).
4. MACS® MultiStand (catalog number 130-042-303).
5. Buffer: prepare a solution 0.5 % Bovine Serum Albumin (BSA,
catalog number A9418) and 2 mM Ethylenediaminetetraacetic
acid (EDTA, catalog number E6758) (Sigma-Aldrich, St.
Louis, MO, USA) in phosphate-buffered saline (PBS, catalog
number 18912-014) (Gibco-BRL, Bethesda, MD, USA),
bring to pH 7.2.
2.3 Flow Cytometry 1. Tubes (or other supports) suitable for your cytometer.
Analysis 2. Antibodies anti-human antigens: CD29 FITC (catalog num-
ber 0791), HLA-DR PE (catalog number 0464), CD105 PE
(catalog number A07414), HLA-ABC FITC (catalog number
IM1838) (Beckman Coulter, Brea, CA, USA), CD44 FITC
(catalog number 560977), 7-aminoactinomycin D (7AAD)
(BD Biosciences, San José, CA, USA), CD73 PE (catalog
number 344004), CD90 FITC (catalog number 328018)
(BioLegend, San Diego, CA, USA).
3 Methods
3.1 Fresh Sample: 1. Centrifuge amniotic fluid sample at 300 × g for 5 min. Remove
Sample Seeding the supernatant making sure not to disturb the cell pellet.
2. Resuspend cells in 3 mL of Chang B + C medium and seed over
a glass coverslip (see Note 4) covering the inside of a
35 mm × 10 mm petri dish.
3. Check daily the cell morphology and change medium after 5–7
days of culture (see Note 5).
4. Once clusters of cells with mesenchymal-like morphology start
to grow (see Note 6 and Fig. 2), immune-selection and subse-
quent expansion should be performed [9].
5. Before proceeding to the next step you should prepare the
Chang Complete medium.
Fig. 2 Morphological aspect of unselected (a, b) and CD117 selected amniotic fluid stem cells (c, d). Images
were taken with phase contrast microscope (scale bars = 100 µm). (a) Morphology of a sample presenting an
epithelial and overconfluent appearance, such samples usually do not give a lot of success for selecting CD117
positive cells. (b) Aspect of a heterogeneous sample with mesenchymal-like cells and also some big-rounded
cells (floating or adherent): choose this type of sample for cells selection. (c) The few cells obtained after
immune-selection and (d) the proliferation of those cells with conservation of the mesenchymal-like morphol-
ogy after culture in expansion medium
3.2 Immune- 1. Take out the supernatant from the petri dish and wash twice
Selection with PBS. Add 500 μL of Trypsin and incubate for 3–5 min at
37 °C. Check at the microscope the detachment of the cells
(see Note 7).
2. Collect cell suspension and add at least 2 mL of α-MEM con-
taining 25 % of FBS. Centrifuge cells at 300 × g for 5 min.
Remove the supernatant making sure not to disturb the cell
pellet and resuspend in 300 μL of buffer.
3. Add 100 μL of FcR Blocking Reagent and 100 μL of CD117
MicroBeads, mix well, and incubate for 15–20 min at 4 °C.
4. Add 2 mL of buffer to the cell suspension and centrifuge at
300 × g for 10 min. Remove the supernatant making sure not
to disturb the cell pellet and resuspend in 500 μL of buffer.
5. Place a MS Column on the Separator attached to the MultiStand
and wash it once with 500 μL of buffer, place a 15 mL tube
under the tip of the column to collect the flow-through (the
negative fraction can be retrieved centrifuging this tube if
needed).
6. Once the first wash has passed through the column put your
cell suspension onto the column.
7. Wash the column three times with 500 μL of buffer.
8. Remove the column from the magnetic support and place it on
a 15 mL tube for collection: add 1 mL of buffer onto the col-
umn and using the plunger immediately flush out the content.
9. Repeat steps 5–8 another time to reduce contamination by
unwanted cells (use one new column).
3.3 Cells Expansion 1. Add 2 mL of buffer to the cell suspension from Subheading 3.2,
step 8 and centrifuge at 300 × g for 10 min. Remove the super-
natant making sure not to disturb the cell pellet and resuspend
in an appropriate volume of expansion medium (see Note 8).
2. We recommend to start expanding selected cells in a well of a
24-multiwell plate and then—before confluence—split them in
other appropriate surfaces to maintain subconfluence (<70–
80 %) (see Note 9). Usually we put around 2 × 104 cells in
100 mm × 20 mm dishes and 1 × 105 cells in 150 mm × 25 mm
dishes [10].
3. Freezing of cells should be performed in FBS (or Horse Serum)
containing 5–10 % DMSO and no less than 5 × 105 cells should
be frozen in a cryotube (expand cells until passage 3 in a
150 mm × 25 mm dish before obtaining enough cells to freeze).
In our experience you can thaw 5 × 105 cells in a 150 mm × 25 mm
dish (thawing in a smaller dish can bring cells directly to con-
fluence) [11].
4 Notes
1. Use only Penicillin and Streptomycin. Other antibiotics can

kill the cells.
2. L-Glutamine is not stable in the expansion medium and there-
fore it should be added again if medium is not consumed after
2 weeks, this does not apply to the Chang B + C because you
add L-Glutamine when you seed your cells.
3. FACS sorting can also be performed on fresh samples, but you
must consider the few cells you are going to start with and see
if it is reasonable (for setting up the instrument and perform-
ing the sorting). Moreover, our experience showed that amnio-
cytes are particularly susceptible to this kind of procedure and
sometimes are not able to survive in culture afterwards.
4. Coverslips must be in glass and no other plastic material, also
they should be sterilized (i.e., using an autoclave) for culture
use.
5. Only adherent cells are of interest: take out the old medium
and put the fresh one over the adherent cells.
6. In Fig. 2 some morphology examples of different samples after
several days in culture. We can distinguish a bad (see Fig. 2a)
from a good morphology (see Fig. 2b) and also the appearance
of the cells just after selection (see Fig. 2c) and after few pas-
sages in culture (see Fig. 2d).
7. For samples of adherent cells coming from the cytogenetic
laboratory start from this point. Epithelial-like cells are difficult
to detach with Trypsin; therefore, you should not wait until all
cells are detached from the coverslip, you should recover your
supernatant when no more mesenchymal-like cells are attached
(otherwise trypsin could damage the cells of interest).
8. In Subheading 3.2, step 8 you can flush the column directly
with the expansion medium (same volume, 1 mL) and put the
resulting cell suspension in culture.
9. CD117 surface expression is reduced after expansion and just
a little portion of the population still express this marker in
culture, the cells present the most common markers of mesen-
chymal stem cells described in literature [12, 13] (see Fig. 3).
CD105
CD29
CD90
CD73
BEFORE SELECTION
HLA-ABC
% of total
HLA-DR
CD44
CD105
CD29
CD90
CD73
AFTER SELECTION
HLA-ABC
% of total
HLA-DR
CD44
CD117
Fig. 3 Representative cytograms of the fresh and expanded (passage 4) cells analyzed for the surface markers
CD29, CD44, CD90, CD73, CD105, HLA-ABC, HLA-DR, and CD117. CD117 surface expression generally dimin-
ishes after expansion; on the contrary, after culture mesenchymal stem cell markers are up-regulated. HLA-
ABC marker in culture is homogeneously expressed, whereas HLA-DR remains negative both in fresh and
expanded CD117 positive cells [14]
References
1. Dobreva MP, Pereira PN, Deprest J et al with potential for therapy. Nat Biotechnol
(2010) On the origin of amniotic stem cells: of 25(1):100–106
mice and men. Int J Dev Biol 54(5):761–777 5. De Coppi P, Callegari A, Chiavegato A et al
2. Pozzobon M, Ghionzoli M, de Coppi P (2010) (2007) Amniotic fluid and bone marrow
ES, iPS, MSC, and AFS cells. Stem cells exploi- derived mesenchymal stem cells can be con-
tation for pediatric surgery: current research verted to smooth muscle cells in the cryo-
and perspective. Pediatr Surg Int 26(1):3–10 injured rat bladder and prevent compensatory
3. Prusa AR, Hengstschlager M (2002) hypertrophy of surviving smooth muscle cells.
Amniotic fluid cells and human stem cell J Urol 177(1):369–376
research: a new connection. Med Sci Monit 6. Piccoli M, Franzin C, Bertin E et al (2012)
8(11):RA253–RA257 Amniotic fluid stem cells restore the muscle cell
4. De Coppi P, Bartsch G Jr, Siddiqui MM et al niche in a HSA-Cre, Smn(F7/F7) mouse
(2007) Isolation of amniotic stem cell lines model. Stem Cells 30(8):1675–1684
7. Ditadi A, de Coppi P, Picone O et al (2009) 11. Janz Fde L, Debes Ade A, Cavaglieri Rde C
Human and murine amniotic fluid c-Kit+ Lin- et al (2012) Evaluation of distinct freezing
cells display hematopoietic activity. Blood methods and cryoprotectants for human amni-
113(17):3953–3960 otic fluid stem cells cryopreservation. J Biomed
8. Cananzi M, Atala A, de Coppi P (2009) Stem Biotechnol 2012:649353
cells derived from amniotic fluid: new poten- 12. Kim J, Lee Y, Kim H et al (2007) Human
tials in regenerative medicine. Reprod Biomed amniotic fluid-derived stem cells have charac-
Online 18(Suppl 1):17–27 teristics of multipotent stem cells. Cell Prolif
9. Klein JD, Fauza DO (2011) Amniotic and pla- 40(1):75–90
cental mesenchymal stem cell isolation and cul- 13. Tsai MS, Hwang SM, Tsai YL et al (2006)
ture. Methods Mol Biol 698:75–88 Clonal amniotic fluid-derived stem cells express
10. Roubelakis MG, Pappa KI, Bitsika V et al characteristics of both mesenchymal and neural
(2007) Molecular and proteomic characteriza- stem cells. Biol Reprod 74(3):545–551
tion of human mesenchymal stem cells derived 14. Abumaree M, Al Jumah M, Pace RA et al
from amniotic fluid: comparison to bone mar- (2012) Immunosuppressive properties of
row mesenchymal stem cells. Stem Cells Dev mesenchymal stem cells. Stem Cell Rev 8(2):
16(6):931–952 375–392
Chapter 17
Hypoxia and Visualization of the Stem Cell Niche

Ali Dalloul
Abstract
It is widely accepted that mammalian stem cells reside in a specialized cellular and a cellular microenvironment
called the niche. The niche contrary to other tissues is characterized by a low partial Oxygen pressure
(ppO2). This microenvironment protects stem cells from deleterious effects of O2 on proteins and DNA,
through the production of reactive oxygen species (ROS). In addition there is now solid evidence that this
physiological hypoxia helps stem cells maintaining their major characteristics: multipotency and ability to
differentiate and migrate from the niche to specialized tissues in order to fulfill the needs of the organism.
Immuno Histological techniques can stain stem cells in situ by specific Abs (such as against CD34 and
CD45 for Hematopoietic Stem Cells HSC). However, a universal marker of hypoxia is Hypoxia-Inducible
Factor-1, HIF-1, which is stabilized by low ppO2 and acts as a transcription factor to regulate a vast array
of genes downstream. HIF-1, together with pimonidazole, a chemical compound interacting with proteins
that are reduced in a hypoxic environment, are bona fide markers of the stem cell niche.
Key words Stem cell niche, Multipotency, Hypoxia, Pimonidazole, Regenerative medicine
1 Introduction
1.1 General Oxygen is a critical component of the stem cell niche which can be
Introduction defined as a specialized microenvironment made of cellular and a
cellular components that integrate systemic and local cues to regulate
the biology of stem cells [1]. As stem cells are vital to the organism,
from a finalistic point of view it is understood that the niche is dedi-
cated to preserving the functions of stem cells [2]. This is achieved
by avoiding metabolic stresses such as those generated by free
oxygen radicals/reactive oxygen species ROS. Furthermore it helps
maintaining long term survival in quiescent state, multipotency,
proliferation and migration capacities of stem cells.
In mammalian tissues, the partial pressure of Oxygen, ppO2
drops progressively after it enters the lungs, from 21 to less than
9 % in most tissues [3] and as low as 2 % in the most hypoxic tissues
and therefore constitute “physiologic normoxia” [4]. The cur-
rently best known niche is the hematopoietic stem cell HSC niche
199
200 Ali Dalloul
which resides in bone cavities adjacent to the endosteum, that is

on the outer region of the medulla, away from the arterial blood
stream [5, 6]. As the diffusion distance of O2 is 150 μm, the esti-
mated ppO2 ranges from 6 % in the sinusoidal cavities to 1 % in
the niches [2]. Hypoxia is also a feature of neurogenic niches in the
brain [7], in brain tumors [8] and several mesenchymal stem cell
MSC niches [9, 10]. Of interest MSC have been shown recently to
reside in the same niche than HSC [11]. It was not totally surprising
therefore that long term cultivation of human bone-marrow MSC
under 5 % ppO2 instead of 21 % ppO2, resulted in enhanced cell
viability and increase in multipotency [12].
It is also inferred from these findings that stem cell progenitors
cultured under low ppO2 would be protected from the harmful
effects of O2, contrary to cells cultured in ambient air with 5 % CO2
as usually done in almost all laboratories. Cultivating the cells in
low ppO2 for regenerative medicine purposes may increase their pro-
liferation efficiency and engraftment capacities upon intravenous
injection. Much less is known however on the effects of hypoxia on
the anti-inflammatory properties of MSC, the potential of which to
treat severe autoimmune disorders has been emphasized during last
decade [13]. Of note, most of our knowledge in this field is about
MSC possibly because these cells need to be passaged in culture for
several weeks to be expanded and are easy to manipulate. In contrast
much less is known about the behavior of embryonic stem cell lines
and of induced pluripotent stem cells iPSC. This may prove of inter-
est as during the time of blastocyst implantation in the uterus where
ppO2 is as low as 2 % [14].
1.2 Biological High ppO2 can be detrimental to stem cells, conversely, hypoxia is
Functions of Hypoxia protective by lowering the local production of ROS. ROS production
is however finely tuned and has also protective effects.
ROS are potentially harmful to stem cells. These anions (O2–)
and hydrogen superoxide H2O2 are detrimental for the lifespan of
HSC, partly through the stimulation of the p38MAPK pathway
[15]. ROS affects genetic stability and their deleterious effects
include DNA damage, aneuploidy, and telomere shortening, all of
which can be prevented in hypoxia [16].
Maintaining quiescence and multipotency is influenced by the
regulation of response to ROS. The FoxO genes [17, 18] are critical
in regulating this response, indeed mice with conditional deletion
of Foxo in HSC, show an increase in cell cycling and apoptosis in
parallel to an increase in ROS species [19]. Critical enzymes for
the production of ROS are NADP oxidase isoforms Nox. Nox are
transmembrane molecules that function as oxygen sensors; they
are produced mainly by differentiated myeloid cells [20] and to a
lesser extent by progenitor cells [21]. Interestingly, in response to
hypoxia, Nox2 induces the production of Matrix Metalloproteinases
MMP, which in turn enhances the migration of stem cells [22].
Hypoxia and Visualization of the Stem Cell Niche 201
This allows HSC to repopulate other niches and possibly MSC to

exert a protective role in ischemic tissues.
Enhancement of migratory capacities: Hypoxic injury results
in the activation of proteolytic enzymes including matrix metallo-
proteinases MMP produced by neutrophils. Extracellular matrix
degradation inhibits adhesion of HSC to the stroma there by helping
their migration to the periphery [19, 22]. MMP degradation, such
as that induced by Granulocyte-colony-stimulating factor GCSF,
helps HSC shedding into the blood stream and has clinical applica-
tions. In contrast, the effect of MMP degradation on the behavior
of MSC is less clear, inasmuch as these cells may home but fail to
engraft into non-hematopoietic tissues and fuse with differentiated
cells instead of differentiating in specialized cells in situ [23].
Several reports pointed however that MSC cultured in low ppO2
expressed more receptors to chemokines (CXCR4, CXCR7 and
CX3CR1) [12, 24], the physiological relevance of this is a matter of
debate, yet it may allow the cells to migrate into (CXCL12/SCF1
and Fractalkine)-producing injured tissues.
Most of the cellular effects on stem cells are mediated by the
transcrfiption factor HIF-1. HIF is the major O2 sensor of hypoxia
in mammalian cells. This heterodimer comprises HIF-1β which is
stable and HIR-1α that is hydroxylized on proline residues by
oxygen-sensitive proline hydroxylases; this allows HIF-1 to be rec-
ognized and ubiquitinated by the E3-unbiquitin ligase family,
tumor suppressor Von Hippel Lindau protein (VHL) under nor-
mal ppO2. Conversely, HIF-1α is stabilized under low ppO2 [25].
This allows the formation of HIF-1 heterodimers which translo-
cate into the nucleus to regulate a vast array of genes. These include
VEGF [26], pyruvate dehydrogenase kinase that allows the cells to
adapt to hypoxia [27]. Most interestingly HIF-1 activates the
Notch-signaling, a major pathway needed for the maintenance in
undifferentiated state [28]. These molecular events are summa-
rized in Fig. 1.
2 Materials
1. Chemicals: pimonidazole (hypoxyprobe, Bioscience Research/

Chemicon, Temeccula, CA), PBS EDTA, Citrate Buffer, MEtOH,
BSA, Triton, Tween, Glycine, Na Citrate, can be purchased
from Sigma Aldrich. OCT and DAPI (Fisher Scientific).
2. Antibodies: for human/rat HIF-1α (Rabbit MAb, Epitomics/
Abcam, ref: 2015-1 at 10 μg/ml), for mouse HIF-1α
(biotin- or HRP-Mouse MAb, Acris, San Diego CA ref:
NB100-131B, 131-H). For Pimonidazole (mouse MAb
Bioscience Research, 20 μg/ml), Antibodies to human or
mouse Nestin (LSBio, Seattle, WA), VEFG (Rabbit anti-
human/mouse, Abcam, Cambridge MA).
202 Ali Dalloul
Low O2 tension HIF-1α

Notch-L
HIF-1β
Notch Inactive PH
Undegraded HIF-1α HRE
+
I Notch
glucose
Survival, multipotency
Lactic acid
LDH
VEGF, CX3CR1, CXCR4, CXCR7
pyruvate
PDK1 PDH
Mitochondrial
respiration
GT, LDH, PDK1
Fig. 1 Molecular action of Hypoxia-activates HIF-1. Under low ppO2, Prolyl Hydroxylase PH, is not activated and
does not tag HIF-1a on pralines for ubiquitination and degradation. HIF-1a dimerizes with HIF-1b to translocate
into the nucleus and interacts with hypoxia-responsive element HRE on the promoters of various genes.
Transcription of chemokine receptor genes to CXCL-12/SDF-1 and CX3CL1/fractalkine, VEGF, genes involved
in multipotency, and genes involved in the inhibition of mitochondrial respiration is activated. HIF-1 interacts
with Notch-ligand(L) to activate the cleavage of Notch. HIF-1 interacts thereafter with intracellular Notch to
activate stemness genes. HIF-1 activates the transcription of glucose transporters, GT, Lactate dehydrogenase
LDH, and Pyruvate kinase dehydrogenase-( PDK-)1. This allows lactic acid to be produced from pyruvate, while
inhibiting pyruvate dehydrogenase and downstream, tricarboxylic (Krebs) cycle, electron transport, ATP and
ROS production. As a result, mitochondrial respiration is inhibited
3 Methods
Current techniques rely on the detection of stem cells and/or mark-

ers of hypoxia, the most reliable of which are hypoxia-inducible
factor 1, and the detection of protein modifications following
hypoxia.
Stem Cell Antigens: paraffin sections of decalcified bone can
be stain with Stem cell markers: At present, MSC can be visualized
by a combination of surface markers, however none of them is
lineage-specific [12] and cannot be used alone for in situ detection.
Human HSC can instead be visualized by double immunostaining

with anti-CD45 and anti-CD34 mAbs. Anti-Nestin Abs to stain
the intermediate filament Nestin, a marker of MSC [11] and
Neural stem cells [29] are useful.
Another strategy involves the detection of HIF1alpha [20] or
Nox2 [21] or VEGF [30] as sensors of hypoxia, by means of Ab
staining. Hypoxia can also be visualized by chemical reagents com-
bined to immunohistochemistry/immunofluorescence IHC/IF:
pimonidazole binds to thiol-containing proteins specifically in
hypoxic cells [31]. This nitroimidazole compound is reduced in an
hypoxic environment and accumulates in hypoxic tissues; it can be
injected iv or taken orally at 0.5 g/m2 and trephine bone marrow
biopsy performed the next day. Mice can also be injected with
60 mg/kg intraperitonealy and bone processed after 3 h and
detected with anti-pimonidazole Ab; [22]. Combining anti-CD34,
anti-CD45, and anti-pimonidazole mAbs should also prove useful
to localize the endosteal HSC niche in humans (see Note 1).
3.1 Immunohisto- 1. Fix the bone with 2 % Paraformaldehyde in phosphate buffer

chemistry saline PBS, for 30 min. For frozen sections, the structure of
the Bone marrow central region can be preserved by direct
flushing of bone cavities with OCT compound.
2. Decalcify with 10 % EDTA for 7 days at 4–8 °C.
3. Embed with Paraffin and dewax by heating in the microwave.
4. Rehydrate in water for 30 min.
5. Antigen retrieval in 10 mM citrate buffer pH 6, at 90 °C for
30 min.
6. Wash twice in PBS at RT for 2 min.
7. Incubate with PBS 50 mM Glycine pH 3.5 for 5 min.
8. Wash in PBS and incubate in PBS 0.3 % Triton X-100 for
15 min.
9. Incubate in methanol 3 % H2O2 for 15 min.
10. Wash 3 times in PBS 0.05 % Tween.
11. Block with 5 % BSA, 0.05 % Triton X-100 in citrate buffer
(0.6 M NaCl, 0.06 M Na-citrate, pH 6.4) + 10 μg/ml mouse
IgG for 60 min RT.
12. Incubate with primary fluorescent or biotinylated Anti-
pimonidazole mAb (Chemicon) 10 μg/ml for 60 min in
humidified atmosphere.
13. Incubate and reveal with a secondary Ab for detection by fluo-
rescence or by IHC. For fluorescence detection, counterstain
with DAPI (see also Note 2).
204 Ali Dalloul
4 Notes
1. IHC is here optimized for the staining of bone marrow cavities.

Soft tissues can optimally be stained on frozen sections. Mince
with scissors to small 1–2 × 2 cm pieces, pre-cool in isopentane,
alternatively cover with OCT-tissue tec (ThermoFisher scien-
tific) in cryoplastic well, hold with flat forceps, and freeze in
nitrogen vapor for 60 s. Store in liquid nitrogen for years or at
−80 °C until needed. Cut in 6 μm sections for optimal IHC or
two-color IF staining.
Before staining, warm at RT for 30 min, fix in ice-cold ace-
tone for 5 min, air-dry for 30 min, wash in PBS.
2. In addition to pimonidazole staining, hypoxic niches may be
stained with Abs against HIF1-a, Nestin, or VEGF and detected
either by IF or by IHC. Nonspecific binding is avoided by serum
block using serum from the same specie as the primary Ab.
We recommend 10 % (rabbit, goat, mouse, or others) serum in
PBS for 30 min RT. Incubate Primary Ab at 4 °C overnight
according to the manufacturer’s instructions for the [c],
although using various dilutions may be needed to optimize
the staining. Serum block is mandatory before incubation with
secondary Ab, as above. Controls include the same steps in
incubations with omission of the primary Ab. Secondary Ab
will then be added according to instructions.
References
1. Scadden DT (2006) The stem cell niche as an 9. Harrison JS, Rameshvar P, Chang V et al
identity of action. Nature 441:1075 (2002) Oxygen saturation in the bone marrow
2. Brahimi-Horn MC, Pouyssegur J (2007) of healthy volunteers. Blood 99:394
Oxygen, a source of life and stress. FEBS Lett 10. Matsumoto A, Matsumoto S, Sowers AL et al
581:3582–3591 (2005) Absolute oxygen tension (p(O(2)) in
3. Eliasson P, Johnsson JI (2010) The hemato- murine fatty and muscle tissue as determined
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Chapter 18
Detection and Isolation of Human Disseminated Tumor

Cells in the Murine Bone Marrow Stem Cell Niche
Yusuke Shiozawa, Russell S. Taichman, and Evan T. Keller
Abstract
The presence of disseminated tumor cells (DTCs) in the bone marrow is associated with poor prognosis of
cancer patients. However, little is known about the biology of DTCs due to lack of relevant animal models.
Here, we describe the methods for detecting and isolating human DTCs from the murine bone marrow
niche by PCR using human Alu sequences and by fluorescence-activated cell sorting and immunohistochem-
istry using anti-HLA antibody. These strategies could be useful for exploring the biology of DTCs.
Key words Disseminated tumor cells, Bone marrow niche, ALU, Human leukocyte antigens,
PCR, Flow cytometry, Animal model, Prostate cancer, Immunohistochemistry
1 Introduction
Dormancy is a critical phenomenon, which affects the long-term

survival of tumor cells [1]. Although a primary treatment for localized
tumor appears to be successful, tumors recur either as local recur-
rence or as distant metastasis years or even decades later. Several
studies have shown that disseminated tumor cells (DTCs) shed
from a primary tumor may lie dormant in distant organs for long
periods of time before they can be activated to form metastases
[2–4]. DTCs are believed to be in a prolonged state of G0 [5],
whereby they are capable of escaping from apoptosis or cytotoxic
insults which target actively dividing cells. However, our under-
standing of how dormancy of DTCs is induced or what leads to
activation of the dormant cells is currently restricted in part due to
the limited number of animal models.
It has been demonstrated that DTCs spread to the bone
marrow using equivalent mechanisms as the process of HSC hom-
ing to the bone marrow [6, 7]. In the marrow, hematopoietic stem
cells (HSCs) are known to stay dormant within the supportive
microenvironment, or niche [8]. The major function of the niche is
207
208 Yusuke Shiozawa et al.
Tumor Inoculation
Harvest Harvest Harvest

Bone Tissues Bone Marrow Cells Long Bones
Deplete Hematopoietic Lineage Cells

Isolate genomic DNA Decalcify and Embed in Paraffin
with Magnetic Beads
Identify PCa Cells by PCR Isolate PCa Cells by FACS Detect PCa Cells by IHC
with ALU sequence with Anti-HLA-ABC Antibodies with Anti-HLA-ABC Antibodies
Fig. 1 Schematic experimental model of human disseminated tumor cells detection in murine marrow. Human
cancer cells are inoculated into immunodeficient animals (e.g., intracardiac injection, intravenous injection,
subcutaneous implantation, intratibial injection). At the termination of the experiment, bone tissues are
harvested. Thereafter, human disseminated tumor cells in the murine tissues are identified by polymerase
chain reaction (PCR), fluorescence-activated cell sorting (FACS), and immunohistochemistry (IHC)
to regulate keep HSC quiescent and protect them from undergoing

apoptosis [8]. Therefore, a hypothesis worth considering is that
the DTCs parasitize the bone marrow niche to become dormant
and facilitate resistance to current therapy. Supporting this notion,
we have recently revealed that disseminated prostate cancer (PCa)
cells target the HSC niche in the marrow and compete for occu-
pancy of the niche with HSCs [9]. By distinguish human cells from
murine tissues it was possible to study the metastatic behavior of
disseminated PCa cells.
To track disseminated human PCa in both murine soft and
skeletal tissues, we have used a xenograft model that we previously
developed [10]. In this model, disseminated human PCa cells
can be detected by real-time PCR (QPCR) using primers designed
to detect human Alu sequences [10]. We are now able to isolate
disseminated human PCa cells from the murine bone marrow by
fluorescence-activated cell sorting (FACS) with an antibody target-
ing human leukocyte antigens (HLA) [9]. Moreover, using anti-
HLA antibody, DTCs are visualized in the marrow under a confocal
microscope. An overview of the method is provided in Fig. 1.
Although this concept cannot be applied for monitoring of tumor
cells in the clinical setting, this technique facilitates exploration of
the microenvironmental effects on tumor progression using in vivo
preclinical models. Furthermore, this method should be applicable
to multiple human tumor cell types when used in various animal
models. The proposed model and strategy sheds light on the
Detection of Disseminated Tumor Cells 209
biology of DTCs and their microenvironment, and could provide

new therapeutic approaches for preventing recurrence after periods
of dormancy.
2 Materials
2.1 DTC Detection 1. DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA, USA).
by PCR 2. ABI PRISM™ 96-Well Optical Reaction Plate with Barcode
(code 128) (Applied Biosystems, Foster City, CA, USA).
3. MicroAmp Optical Adhesive Film Kit (Applied Biosystems).
4. TaqMan® Universal PCR Master Mix (Applied Biosystems).
5. RNase-Free Water (QIAGEN).
6. TaqMan® Gene Expression Assay (murine β-actin) (Applied
Biosystems).
7. ALU Primer Mix (Applied Biosystems): 5 μl of TaqMan®
TAMRA™ Probe (100 μM) (5′-FAM-ATT AGC CGG GCG
TGG TGG CG-TAMRA-3′), 10 μl of ALU forward primer
(50 μM) (5′-CAT GGT GAA ACC CCG TCT CTA-3′), 10 μl
of reverse primer (50 μM) (5′-GCC TCA GCC TCC CGA
GTA G-3′), 75 μl of RNase-free water (see Note 1).
8. ABI PRISM® 7700 Sequence Detection System (Applied
Biosystems).
2.2 DTC Isolation 1. Flow Cytometry Staining Buffer: Dulbecco’s Phosphate-

by FACS Buffered Saline (DPBS) (Invitrogen, Grand Island, NY, USA),
2 % Fetal Bovine Serum (FBS) (Invitrogen).
2. BD Falcon™ 100 mm Cell Culture Dish, tissue-culture treated
polystyrene (BD Biosciences, San Jose, CA, USA).
3. BD 3 ml syringe Luer-Lok Tip (BD, Franklin Lakes, NJ, USA).
4. BD Regular Bevel Needles (25 G × 5/8 in.) (BD).
5. 15 and 50 ml high-clarity polypropylene conical centrifuge
tube (BD Biosciences).
6. Fisherbrand® Cell Strainers (40 μm Nylon Mesh) (Fisher
Scientific, Hanover Park, IL, USA).
7. 12 × 75 mm, 5 ml polystyrene round bottom test tube (BD
Biosciences).
8. Lineage Cell Depletion Kit (Miltenyi Biotec Inc., Auburn,
CA, USA).
9. autoMACS™ Separator (Miltenyi Biotec Inc.).
10. FITC anti-human HLA-A,B,C Antibody (BioLegend, San
Diego, CA).
11. FACSAria II cell sorter (BD Biosciences).
2.3 DTC Detection 1. 10 % neutral buffered formalin solution (Sigma-Aldrich, St.

by IHC Louis, MO, USA).
2. Tissue-Tek® Mega-Cassette® System (Sakura Finetek USA.,
Inc., Torrance, CA, USA).
3. Decalcification Solution: 10 % EDTA (Thermo Fisher
Scientific, Fremont, CA, USA) in ddH2O, pH 7.38. Store at
room temperature.
4. Xylene (Thermo Fisher Scientific).
5. 100, 90, 70, 50 % Ethanol.
6. Pepsin solution (Thermo Fisher Scientific).
7. PBT solution: 0.2 % Triton X-100 (Thermo Fisher Scientific)
in DPBS. Store at room temperature.
8. Image-iT™ FX Signal Enhancer (Invitrogen).
9. Purified anti-human HLA-A,B,C Antibody (BioLegend).
10. Zenon labeling kit (Invitrogen).
11. Bovine Serum Albumin (BSA) (Sigma-Aldrich).
12. ProLong® Gold antifade reagent with DAPI (Invitrogen).
13. Olympus FV500 confocal microscope (Olympus Imaging
America Inc., Center Valley, PA, USA).
3 Methods
In our study, tumor inoculation into immunodeficient mice is

usually performed by subcutaneous implantation (2 × 105 PCa
cells/animal) for 3 weeks [10] or by intracardiac injection (1 × 106
PCa cells/animal) for 24 h [9] (see Note 2).
3.1 DTC Detection 1. Create a standard curve comparing total mouse cells to ß-actin
by PCR cycles: Count and separate 1 × 107 mouse cells into one tube,
dilute 1:10 to make a total of eight standards ranging from
1 × 107 cells to 1 cell.
2. Extract genomic DNA using the DNeasy Blood & Tissue kit.
3. Plate samples in a PCR plate: 15 μl TaqMan Master Mix;
12.5 μl H2O; 1.5 μl mouse actin primer; 1 μl DNA sample.
4. Run the 2nd step PCR reaction for 40 cycles (95 °C for 15 s
and 60 °C 1 min) after an initial single cycle of 50 °C for 2 min
and 95 °C for 10 min using an ABI PRISM 7700 instrument.
5. Note the threshold cycle.
6. Make the graph using Excel by plotting cell number on the
x-axis and cycle number on the y-axis on a scatter plot, and
then add a trendline (see Note 3).
7. Find the inverse of the equation of the trendline shown on the

graph. This inverse equation will be used to determine how
many mouse cells (the x-value) are in the sample by using the
actin cycle number determined by the PCR (the y-value).
8. Create a standard curve comparing total human cancer cells to
ALU cycles: Repeat steps 1–7 using human cancer cells and
ALU primer mix (15 μl TaqMan Master Mix, 13 μl H2O; 1 μl
ALU primer mix; 1 μl DNA sample) (see Note 4).
9. Determine the amount of mouse cells in experimental samples:
After tumor inoculation, harvest murine organ/tissue and
extract genomic DNA using the DNeasy Blood & Tissue kit
(see Note 5).
10. Run PCR (15 μl TaqMan Master Mix; 12.5 μl H2O; 1.5 μl
mouse actin primer; 1 μl DNA sample).
11. Determine the amount of mouse cells in each organ/tissue
sample using the inverse equation created in step 7.
12. Determine the amount of human cancer cells in experimental
samples: Repeat steps 9–11 using the inverse equation created
in step 8.
13. Normalize the number of total human cancer cells (obtained
from step 12) against the number of total mouse cells (obtained
from step 11).
3.2 DTC Isolation 1. Harvest murine long bones.

by FACS 2. Strip off surrounding muscle tissue thoroughly and carefully.
3. Cut both ends of the bone.
4. Flush out the bone marrow cells with buffer using a syringe
and a 25G needle and collect the cells in the 100 mm cell cul-
ture dish.
5. Filter the cells through 40 μm nylon mesh cell strainers to
obtain single-cell suspensions in the 50 ml conical tube and
count the cells number.
6. Wash the cells with DPBS, centrifuge at 2,250 g at 4 °C for
5 min, aspirate supernatant.
7. Resuspend the cell with 40 μl/107 cells of staining buffer and
transfer into 15 ml conical tube (see Note 6).
8. Add 10 μl/107 cells of Biotin-Antibody Cocktail from the
Lineage Cell Depletion Kit.
9. Incubate at 4 °C for 10 min in the dark and vortex the sample
every 5 min.
10. Wash as in step 6.
11. Resuspend the cell with 30 μl/107 cells of staining buffer.
12. Add 20 μl/107 cells of Anti-Biotin MicroBeads from the

Lineage Cell Depletion Kit.
every 5 min.
15. Resuspend the cell with 1 ml of staining buffer (see Note 7).
16. Collect the lineage negative population with autoMACS™
Separator (see Note 8).
18. Resuspend the cell with 100 μl of staining buffer and transfer
into 5 ml round bottom test tube.
19. Add 20 μl of FITC anti-human HLA-A,B,C Antibody
(see Note 9).
every 10 min.
22. Resuspend the cell with 400 μl of staining buffer.
23. Analyze or isolate the HLA-A,B,C positive cells by flow
cytometry.
3.3 DTC Detection 1. Harvest murine long bones.

by IHC 2. Strip off surrounding muscle tissue thoroughly and carefully.
3. Fix bones with 10 % neutral buffered formalin solution with
agitation overnight at room temperature (see Note 10).
4. Put bones into a tissue cassette.
5. Transfer bones into decalcification solution for 21 days at 4 °C
(see Note 11) with agitation with frequent solution changes
(see Note 12).
6. Embed decalcified bones in paraffin and sectioned at 7 μm
thickness for histological examination (see Note 13).
7. De-paraffin the paraffin prepared slide in xylene for 10 min ×2.
8. Rehydrate the slide in 100, 90, 70, 50 % Ethanol for 5 min in
each step, then DPBS for 10 min.
9. Retrieve the section by dropping pepsin solution over the
section at 37 °C for 15 min in a wet chamber.
10. Wash the section with DPBS for 5 min.
11. Permeabilize the section by incubating the slides in PBT solution
for 10 min.
12. Block the section with Image-iT™ FX Signal Enhancer at room
temperature for 30 min (see Note 14).
13. Prepare Zenon complex staining solution using Zenon labeling
kit: First dilute 1 μg of Purified anti-human HLA-A,B,C
Fig. 2 Representative bone marrow histology following establishment of disseminated tumor cells. Intracardiac
injection of PCa cells (PC3) into severe combined immunodeficiency (SCID) mice was performed. Twenty-four
hours later, the long bones were harvested and human disseminated tumor cells in murine marrows were
visualized by immunofluorescent imaging with anti-HLA-ABC antibodies. Vehicle injection (saline) served as a
negative control. Original magnification 60× Zoom2. Bar = 20 µm
Antibody in 20 μl of DPBS with 0.1 % BSA, add 5 μl of Zenon®

labeling reagent from Zenon labeling kit, incubate at room
temperature for 10 min, add 5 μl of Zenon® blocking reagent
from the kit, then incubate at room temperature for 5 min
(see Note 15).
14. Incubate the section with Zenon complex staining solution
(see Note 16) in a wet chamber at room temperature for 2 h
(see Note 17).
15. Wash the slides in PBT solution in Coplin jar with gentle shaking
for 5 min ×2–3.
16. Post-fix the section with 10 % neutral buffered formalin solution
at room temperature for 10 min.
17. Mount the section in the ProLong® Gold antifade reagent with
DAPI with the cover glass.
18. Visualized HLA-A,B,C positive cells under a confocal micro-
scope (Fig. 2).
4 Notes
1. This ratio of probe to primers optimizes the PCR signal, and

also allows for a good amount of separation between sample
and contamination signals.
2. Any xenograft models of human cells in immunodeficient
animals can be used as a tumor inoculation.
3. The closer the R2 value is to 1, the better the model fits the
data. If necessary, delete points that lie too far away from
the current trendline to optimize the trendline. However, do

not use the line obtained from less than 3 or 4 plots.
4. To prevent the possible DNA contamination from the air from
the laboratory area, it is important to wear disposable gloves and
face masks. Preferably, sample preparation can be performed
in a sterilized area, such as laminar flow hood with UV light.
In addition, including negative control reactions (e.g., H2O) is
essential to detect contamination.
5. Harvested organs/tissues can be stored at −80 °C until
genomic DNA is extracted.
6. Steps 7–17 can be skipped if the lineage depletion is not nec-
essary for analysis or sorting.
7. Since the cell clumps will cause the clog in the system, the clumps
should be removed before applying magnetic separation.
8. Alternatively, the manual separation can be performed using
MACS® Column.
9. Since each cells express different type of HLA on their surface, it
is important to find out which HLA the cells that are analyzed
express prior to the experiment.
10. Alternative fixation methods can be applied based on the antigen
retrieval techniques.
11. We find that this is the best decalcification duration to demin-
eralize the bone tissues with preservation of both bone and soft
tissue structures.
12. Alternative decalcification methods can be applied based on
the antigen retrieval techniques.
13. Decalcified bones can be stored in 70 % ethanol until bones are
processed for paraffin embedding.
14. Alternative block reagent can be 5–10 % heated normal goat
serum.
15. Complex should be freshly made and applied within 30 min.
16. Working concentration of complex is usually 3–5 times
higher than that of the primary antibody for standard
immunohistochemistry.
17. Do not incubate any longer, otherwise the Zenon Fab fragment
can dissociate from the primary antibody.
Acknowledgments
We thank Elisabeth A. Pedersen, Aaron M. Havens, Jingcheng

Wang (University of Michigan, Ann Arbor, Michigan, USA) for
technical and logistical support. This work is directly supported by
the National Cancer Institute (CA093900, E.T.K. and R.S.T.,

CA163124, Y.S. and R.S.T.), the Department of Defense (Y.S. and
R.S.T.), and the Prostate Cancer Foundation (Y.S. and R.S.T.).
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(2009) Disseminated tumor cells in prostate 8. Taichman RS (2005) Blood and bone: two
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without evidence of disease predicts biochemi- the hematopoietic stem-cell niche. Blood 105:
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Requirement of KISS1 secretion for multiple (2011) Human prostate cancer metastases target
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99:309–321 121:1298–1312
5. Holmgren L, O’Reilly MS, Folkman J (1995) 10. Havens AM, Pedersen EA, Shiozawa Y et al
Dormancy of micrometastases: balanced prolif- (2008) An in vivo mouse model for human
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Chapter 19
Identification and Separation of Normal Hematopoietic

Stem Cells and Leukemia Stem Cells from Patients
with Acute Myeloid Leukemia
Van T. Hoang, Isabel Hoffmann, Karina Borowski,
Abraham Zepeda-Moreno, Dan Ran, Eike C. Buss,
Patrick Wuchter, Volker Eckstein, and Anthony D. Ho
Abstract
Mounting evidences indicate that leukemic cells in patients with acute myeloid leukemia (AML) are
derived from leukemia stem cells (LSC). In analogy to normal hematopoietic stem cells (HSC), LSC
remain mostly dormant and are hence resistant to conventional chemotherapy. Residual, physiological
HSC exist alongside with LSC, with heterogeneous dominance of LSC over HSC in individual patients.
We have devised a flow cytometric method for the identification and separation of these two stem cell
populations based on surface antigen markers such as CD34, CD38, lineage aberrant markers, and alde-
hyde dehydrogenase (ALDH) enzyme activity.
Key words Acute myeloid leukemia, Leukemia stem cells, Hematopoietic stem cells, Flow cytometry,
FACS, Aldehyde dehydrogenase, Aberrant markers
Abbreviations
ALDH Aldehyde dehydrogenase
AM Aberrant marker
AML Acute myeloid leukemia
APC Allophycocyanin
FSC Forward scatter
FACS Fluorescence-activated cell sorting
FITC Fluorescein
HSC Hematopoietic stem cell
LSC Leukemia stem cells
MNC Mononuclear cells
NOD/SCIDNOD/SCID Non-Obese Diabetic/Severe Combined Immune Deficiency
NSG NOD/SCID/interleukin 2 receptor gammanull
217
218 Van T. Hoang et al.
PE Phycoerythrin
PI Propidium iodide
SSC Side scatter
1 Introduction
Flow cytometry has become an essential tool in diagnostics for

visualization of cellular physical and chemical characteristics, e.g.
size, granularity, surface and intracellular protein expression, as
well as DNA content. Advantages of this method include the abil-
ity to quantitatively analyze a large number of events in a short
time, and the characterization of co-expressions of several mole-
cules simultaneously in individual cells. It allows the identification
and separation of rare cell compartments within a heterogeneous
population such as HSC in the hematopoietic system. In this study,
we have devised a method for the isolation of rare stem cells in
patients with AML and their separation into leukemia (LSC) and
normal hematopoietic stem cells (HSC) for further functional
characterization. This method can be applied for samples at the
time of diagnosis as well as of follow-up examination for detection
of resistant LSC, which might be responsible for relapse.
AML results in accumulation of nonfunctional leukemic cells in
the bone marrow and blood, with subsequently infiltration in other
organs such as the spleen and the liver. The disease incidence
increases with life span and the median age of patients with AML
was 65 years [1]. Although complete remissions can be induced in
70–80 % of patients younger than 60 years and in approximately
50 % of older patients, many who initially respond still suffer from
relapse, such that, without allogeneic transplantation, the 5 year-
survival rates are approximately 30 % in the younger and 10 % in the
older age group [2]. The persistence of LSC is probably responsible
for recurrences, as the LSC are quiescent, well protected within
the niche, and therefore chemo- and apoptosis-resistant [3–5].
LSC were reported to reside in the CD34+CD38− cell popula-
tion, since these cells, but not the CD34+CD38+ cells were able to
engraft and induce leukemia in immunodeficient NOD/SCID
mouse model [6, 7]. Recent studies using an even more immuno-
deficient NOD/SCID/IL2Rγ−/−(NSG) mouse model have shown
that LSC can also be found in other subpopulations such as
CD34+CD38+ and CD34− in certain cases [8, 9]. In patients with
mutated nucleophosmin, one of the most frequently mutated
genes in AML, a low frequency of CD34+ cells was observed and
this was associated with a normal origin of these cells [10]. In these
cases, LSC were found in the CD34− fraction and no specific mark-
ers for their enrichment could be identified so far. In this protocol
we will focus mainly on patients with CD34+ AML who presented
with more than 20 % CD34+ cells.
Separation of Normal Hematopoietic and Leukemia Stem Cells in AML 219
Most authors reported that HSC and LSC share the same
CD34+CD38− phenotype. Thus in patients with AML, this subset
consists of a mixture of both of normal HSC and LSC compart-
ments. Other authors have suggested that several surface markers
were differentially expressed on LSC as compared to HSC. These
included CD90 [11], CD123 [12], aberrant markers (AM) [13],
CD96 [14], CD47 [15] and Tim3 [16]. With the exception of
CD90, which is expressed at a higher level in HSC than in LSC,
all the other markers have been reported to be up-regulated in LSC.
AM can be lymphoid lineage markers, e.g., CD2, CD5, CD7,
CD19, CD22 and CD56, or differentiated myeloid marker, e.g.,
CD11b, which are absent in normal HSC, but are aberrantly
expressed in LSC and immature leukemic blasts. They have been
used for detection of minimal residual disease in the routine diag-
nostics and in combination with CD34 and CD38 for an enrich-
ment of LSC and HSC [13]. The disadvantages of the technique are
that it requires an individual strategy for each patient, a large panel
of antibodies and the quality of the enrichment might vary between
different markers. Other candidates for the separation of HSC and
LSC are the recently published markers CD47 and Tim3. However,
CD47 was expressed unexpectedly high in all hematopoietic cells
including HSC in healthy bone marrow and Tim3 was upregulated
only in some AML samples in our analysis.
In this protocol, we describe a method using the enzyme alde-
hyde dehydrogenase 1 (ALDH) as a marker for the detection and
separation of HSC and LSC from the same patient with AML at
the time of diagnosis as well as in follow-up studies. ALDH belongs
to the ALDH super-family, which is expressed in many tissues and
is responsible for the oxidation of aldehydes to corresponding
carboxylic acids. These enzymes are involved in many metabolic
processes, e.g., alcohols, amino acids, vitamins, steroids, and lipids,
as well as in the detoxification of drug-induced aldehyde substrates
leading to resistance of cells [17]. Moreover, ALDH has been
shown to be expressed at a high level by primitive stem and pro-
genitor cells in the bone marrow [18, 19], brain [20], breast [21],
and in several cancers such as AML [19, 22, 23], breast [21], colon
[24] and prostate cancer [25]. In the bone marrow of both healthy
donors and patients with AML, cells with high activity of ALDH
(ALDHbright) contained significantly higher proportions of long-
term colony initiating cells and engrafted better in mouse models
than their counterparts [19, 22, 23]. However, its expression in
AML is more heterogeneous. In the majority of patients, ALDHbright
cells are very rare. These cells have been demonstrated to contain
residual normal HSC and could be distinguished from CD34+CD38−
LSC that exhibited lower level of ALDH (ALDHdim) [26]. In this
protocol, we demonstrate the identification of the ALDHbright cells
in AML patients and the analysis of their co-expression of AM,
which provides additional important information supporting the

notion that cells expressing high levels of ALDH but without
co-expression of AM are of normal origin. Finally, we introduce a
flow cytometric technique for the purification of HSC and LSC
from the same sample using CD34, CD38 and ALDH. These
sorted cells can be used for many downstream applications, e.g.,
cell culture, transplantation into xenografts, cellular and molecular
analyses.
2 Materials
2.1 Patient Samples Bone marrow aspirates of patients with AML are collected in hepa-
rin. Samples should be stored at 4 °C and processed as soon as
possible. Frozen mononuclear cells can also be used.
2.2 Materials Biocoll separation solution (1.077 density, Biochrom AG, Berlin).
for Gradient PBS (PAA Laboratories GmbH, Cölbe).
Separation of
EDTA (PAA Laboratories GmbH, Cölbe).
Mononuclear
Cell (MNC) FBS (PAA Laboratories GmbH, Cölbe).
Cell preparation buffer: PBS added with 1 % FBS and 2 mM EDTA.
Store the buffer at 4 °C.
Türk’s solution (Merck, Damstadt).
Or Trypan blue (FulkaChemi AG, Taufkirchen).
Hemocytometer (Neubauer counting chamber).
BD Pharm Lyse™ reagent (BD Bioscience, Heidelberg).
2.3 Materials and FACS snap-cap tubes (12 × 75 mm, polystyrene round-bottom
Antibodies for Flow tubes) (BD Bioscience, Heidelberg).
Cytometry Aldefluor kit (Stem Cell Technologies, Vancouver, BC,
Canada).
Antibodies:
CD38 Phycoerythrin (PE) (clone HB7, BD Bioscience, Heidelberg).
CD7 PE (BD Bioscience, Heidelberg).
CD11b PE (BD Bioscience, Heidelberg).
CD15 PE (BD Bioscience, Heidelberg).
CD19 PE (clone SJ25C1, BD Bioscience, Heidelberg).
CD56 PE (clone MY31, BD Bioscience, Heidelberg).
CD34 Allophycocyanin (APC) (clone 8G12, BD Bioscience,
Heidelberg).
CD45 APC-H7 (clone 2D1, BD Pharmingen, Heidelberg).
Propidium iodide (PI, BD Bioscience, Heidelberg) in order to
exclude dead cells.
2.4 Flow Cytometry A FACS sorter that is suitable for Fluorescein (FITC), PE, PI, APC
and APC-H7. The immunophenotyping can be done using a
FACS analyzer. We used a FACScan (BD Bioscience, Heidelberg),
supplemented with a Rainbow laser (Cytek Flow Cytometry
Products, California, USA), which supports the measurement of
APC and APC-H7 fluorochromes, and a FACSAria II sorter (BD
Bioscience, Heidelberg).
3 Methods
3.1 Patient Samples Bone marrow aspirates should be obtained from patients diag-
nosed with AML after informed consent and with the approval of
local ethics committees. Immunophenotyping and other diagnos-
tic analysis can be carried out in the routine diagnostics for the
expression of AM such as CD2, CD7, CD11b, CD15, CD19,
CD22, and CD56 on blasts and CD34+ cells. Candidates for AM
were chosen for individual patient if they are expressed on more
than 50 % of CD34+ cells or more than 50 % of blasts (see Note 1).
3.2 Isolation of MNC 1. Mix 5–20 ml of bone marrow aspirates with PBS to a total
by Ficoll Gradient volume of 35 ml in a 50 ml conical tube.
Centrifugation 2. Place 15 ml of Biocoll separation solution into another 50 ml
conical tube.
3. Carefully overlay the Biocoll separation solution with the bone
marrow/PBS mixture.
4. Centrifuge the tube at 950 × g for 20 min at room temperature
without brake.
5. Carefully transfer the mononuclear cell layer at the interphase to a
new 50 ml conical tube. These cells contain mainly lymphocytes,
monocytes, thrombocytes, blood stem and progenitor cells.
6. Fill the tube with cell preparation buffer, centrifuge at 500 × g
for 5 min with brake, and remove the supernatant.
7. Repeat step 6.
8. Mix a small amount of cells with Türk’s solution and count
them with the Neubauer chamber. White blood cells should be
stained in blue in the Türk’s solution (see Note 2).
3.3 Erythrocyte Lysis After density gradient centrifugation, if the cell preparation still
contains a high number of erythrocytes, an erythrocyte lysis step
should be performed. An erythrocyte to leukocyte ratio higher
than 2:1 might disturb the ALDH staining.
1. Suspend the cell in 500 μl cell preparation buffer.
2. Add 5 ml of 1× BD Pharm Lyse™ reagent to the cell suspension.
3. Incubate 3 min at room temperature.
4. Fill the tube with cell preparation buffer and centrifuge at

500 × g for 5 min with brake, and remove the supernatant.
5. Repeat step 4.
6. Count the cells.
3.4 ALDH Staining Aldefluor substrate is activated, aliquoted, and stored at −20 °C
according to the manufacturer’s instructions. The following proto-
col is adapted from the manufacturer’s instructions for staining a
large number of cells.
1. Adjust sample to a concentration of 1 × 106 to 1 × 107 cells/ml
with Aldefluor assay buffer (see Note 3).
2. Label four FACS tubes for the unstained control (autofluores-
cence), the DEAB control, the ALDH compensation control
and the test tube.
3. Place 100 μl of cells in the unstained control and the rest in the
test tube.
4. Add 2 μl DEAB into the DEAB control tube (see Note 4).
5. Add 5 μl of Aldefluor substrate per 1 ml reaction to the test
tube, mix well, and immediately transfer 100 μl of this mixture
into the DEAB control tube (see Note 5).
6. Incubate the DEAB control and the test tube in water bad at
37 °C for 30 min.
7. Place 100 μl of cells from the test tube in the ALDH compen-
sation control tube.
8. Add 200 μl cold Aldefluor assay buffer to the DEAB and
ALDH control tubes to wash the cells.
9. Centrifuge all the tubes at 500 × g for 5 min and remove
supernatant.
10. Resuspend cells in the control tubes with 200 μl Aldefluor
assay buffer and place on ice or at 4 °C in the dark until FACS
measurement.
11. Cells in the test tube are resuspended in 50 μl Aldefluor assay
buffer and stained with surface markers follows.
3.5 Staining with The emission spectrum of the Aldefluor fluorescent product overlaps
Surface Markers with that of FITC and is compatible with antibodies conjugated to
other fluorochromes. Compensation controls have to be prepared
using cells stained with single fluorochromes, their isotype controls
and/or fluorescence minus one fluorochrome.
1. Label and place 105 cells into each compensation control tube.
2. Wash cells with cell preparation buffer (500 × g, 5 min).
3. Remove supernatant and suspend cells in 50 μl cell preparation
buffer.
4. Add appropriate amount of antibodies to the tubes.
Table 1
The excitation and emission of the fluorochromes
Fluorescence Excitation Excitation Emission

Fluorochrome emission color max (nm) laser line (nm) max (nm)
FITC or Aldefluor Green 494 488 519
substrate
PE Yellow 496, 564 488 578
PI Orange 351, 535 488, 532, 561 617
APC Red 650 633, 635, 640 660
APC-H7 Infrared 650 633, 635, 640 785
It is recommended to perform an antibody titration. We

stain up to 106 cells with 2 µl PE antibodies (CD7, CD11b,
CD15, CD19, CD22, CD38, and CD56), 0.5 µl CD34 APC
and 1 µl CD45 APC-H7 in 50 µl reaction for the analysis. For
the sorting by FACS, we use 10 µl PE antibodies, 2.5 µl CD34
APC and 5 µl CD45 APC-H7 in 50 µl reaction for 107 cells.
Test sample:
ALDH AM CD34 CD45
Sort sample:
ALDH CD38 CD34 CD45
5. Incubate at 4 °C for 30 min in the dark.

6. Wash cells with 500 μl cold Aldefluor assay buffer for the
ALDH stained tubes and cell preparation buffer for the other
tubes (500 × g, 5 min).
7. Suspend cells in cold Aldefluor assay buffer at the concentra-
tion of 106–107 cells/ml for the ALDH stained tubes or cell
preparation buffer for the control tubes. Keep cells on ice or at
4 °C in the dark until measurement.
3.6 Instrument The excitation and emission of the fluorochromes are indicated
Setting and Data in the Table 1 (see Note 6). The instrument setting can vary
Acquisition depending on the flow cytometry system that is provided in your
department. We use the FACScan analyzer and the FACSAriaII
sorter with the CellQuest and FACSDiva software provided by the
manufacturer.
1. Prepare an acquisition template (mask) (Fig. 1).
(a) Create a forward scatter (FSC) vs. side scatter (SSC) dot
plot and a region R1 that encompass MNC (Fig. 1a).
(b) Create a FSC vs. PI dot plot gated on R1 and a region R2
displaying PI negative cells that means living cells (Fig. 1b).
Fig. 1 Data acquisition and gating strategies for ALDHbright, ALDHdim and surface marker expressing cells.
The mononucleated cells (MNC) are gated in the region R1 (a). Dead cells are excluded by PI. The R2 gate
includes living cells from R1 (b) and these are displayed in pink in the following plots. DEAB, an ALDH inhibitor,
is used as a control of the ALDH staining (c). ALDH level in MNC is observed in a dot plot (d) and in a density
plot (e). Gate R3 marks ALDHbright cells (R3), which have a high ALDH level and low SSC. They are absent in
the DEAB control tube. ALDHdim cells show an intermediate level of ALDH and are gated in the region R4. The
ALDHbright cells display low FSC and low SSC (f). Expression of the aberrant marker CD56 and the stem/
progenitor cell marker CD34 is demonstrated in (g) and (h), in which the positive cell fractions are gated as R5
(g) and R6 (h). R7 is the gate of blasts, which are characterized by CD45dimSSClow (i). Spectral overlaps were
compensated as demonstrated in k–m
(c) Create a SSC vs. FITC (or FL-1) dot plot gated on R2 for
the detection of ALDH (Fig. 1c, d, see Note 7).
(d) Create dot plots of FSC vs. PE (or FL-2), APC (or FL-4),
and APC-H7 (or FL-5) vs. SSC gated on R2 for the detec-
tion of appropriated fluorochromes (Fig. 1g–i).
(e) Create dot plots for compensation of spectral overlap
(Fig. 1k–m).
2. Acquisition of data.
(a) Place the non-stained tube in the cytometer, adjust FSC,
SSC voltages and gain to center the MNC within the plot
and the R1 gate (Fig. 1a).
Fig. 2 Analysis of the aberrant markers (AM) expression in AML blasts and in the ALDHbright cell population.
The ALDHbright cells are gated in R3 with a frequency of 0.53 %. These cells express a very low level of AM
compared to the leukemic blasts suggesting their normal origin
(b) Place the PI-staining tube in the cytometer, adjust FL-3

voltage so that PI negative cells are found within the gate
R2 (Fig. 1b).
(c) Adjust the FL-1 voltage using DEAB and ALDH tubes
due to the staining in the test tubes. Compensate spectral
overlap of Aldefluor dye to other channels.
(d) Adjust FL-2, FL-4 and FL-5 voltages and compensate
spectral overlap.
(e) Measure the ALDH, AM, CD34 and CD45 test tube
after adding of PI.
3.7 Data Analysis 1. Add regions R3 and R4 that gate on the ALDHbright and
ALDHdim cells in the ALDH versus SSC plot, respectively.
ALDHbright cells (R3) are characterized as a population with
high ALDH level and low SSC and they are not observed in
the DEAB control tube. ALDHdim cells have an immediate
level of ALDH and can be distinguished from ALDHneg cells,
which are mostly erythrocytes (Fig. 1c–e, see Note 8).
2. Check the FSC/SSC pattern of the ALDHbright cells. They
should have low FSC and low SSC indicating their small size
and low granularity (Fig. 1f).
3. Create a region R5 of AM-expressing cells in the FL-2 vs. FSC
plot (Fig. 1g). Display the frequency of R5 within R2.
4. Create a region R6 of CD34+ cells in the FL-2 vs. FSC plot
(Fig. 1h). Display the frequency of R6 within R2.
5. Create a region R7 of CD45dimSSClow leukemic blasts in the
CD45 vs. SSC plot (Fig. 1i).
6. Display blasts (R7) and ALDHbright cells (R3) in CD34 vs. AM
dot plots and add the same quadrant gate in all plots to sepa-
rate the populations based on CD34 and AM expression
(Fig. 2). Show statistics of the quadrant gate for each plot.
Fig. 3 Separation of HSC and LSC based on ALDH level within the CD34+CD38− population. The ALDHbright and
ALDHdim cell populations are identified as R3 and R4 as mentioned before (a and b). CD34+CD38− cells are
marked by the gate R10 (c). Their ALDH levels are demonstrated with the percentages of CD34+CD38−ALDHdim
LSC and CD34+CD38−ALDHbright HSC (d). In the sample 1, CD34 is expressed in a large part of MNC and
CD34+CD38− cells consist mostly of LSC with a minority of residual HSC. The sample 2 is a typical example for
the CD34− AML group, in which leukemic cells have been shown to reside within the CD34− population,
whereas CD34+ cells define normal HSC and their progenitors. In accordance with this observation, the
CD34+CD38− cells of our sample expressed highly ALDH and were found exclusively within the R3 gate and
represent normal HSC
7. In the majority of AML samples, only rare events of ALDHbright

cells can be detected. The ALDHbright cells form a defined pop-
ulation, clearly distinguishable from the ALDHdim population.
These cells do not or only a very small part of them express
aberrant markers indicating that they are normal HSC (Fig. 2).
Therefore, ALDH can be used for the isolation of HSC and
LSC in the next steps (see Note 9).
3.8 Sorting of HSC 1. Repeat the instrument settings for ALDH, CD38 PE, CD34
and LSC in ALDH− AML APC, and CD45 APC-H7 as described for the ALDH, AM,
Samples by FACS CD34, and CD45 test tube.
2. ALDHbright and ALDHdim cells are gated in the region R3 and
R4 as described before (Fig. 3a, b).
3. Create a CD34 vs. CD38 dot plot. Add a region R10 of
CD34+CD38−/dim cells (Fig. 3c).
4. Display R10 in an ALDH vs. SSC dot plot. Copy region R3
and R4 to this plot to gate CD34+CD38−ALDHbright and
CD34+CD38−ALDHdim cell population (Fig. 3d).
5. Sort CD34+CD38−ALDHbright HSC and CD34+CD38−
ALDHdim LSC in sterile FACS tubes containing IMDM
Fig. 4 FISH analysis for CD34+ALDHbright cells (a) and MNC (b) of an AML sample with a PML/RARA t(15;17)
(q24;q21) specific DNA probe (Kreatech Diagnostics) for detection of a deleted chromosome 17. Nuclei are
stained with DAPI in blue. The probes of chromosome 15 and 17 are labeled in green and red, respectively.
One red signal in the nucleus indicates a deletion of the chromosome 17. CD34+ALDHbright cells representing
normal HSC in this case are free of this deletion (a), which is detected in MNC that mainly consist of leukemic
blasts (b)
supplemented with 1 % FBS and 2 mM EDTA (see Note 10).

The sorted cells can be used for in vitro cell culture, in vivo xeno-
transplantation, FISH, and molecular analysis. Fig. 4 demon-
strates that CD34+ALDHbright cells were indeed free of the
genetic mutation which was detected in the unsorted sample.
6. Reanalyze the sorted cells to determine the purity of the sort.
4 Notes
1. In case the expression of an AM is between 30 and 50 %, we

recommend checking its expression within the CD34+CD38−
population. If more than 50 % of these cells display the marker,
it can be used for the further analysis. On the other hand, if
more than one AM are observed in a sample. The most strongly
expressed marker can be chosen for the study.
2. Trypan blue can also be used, dead cells are then dyed blue, for
the counting of viable cells. It is especially recommended for
frozen samples, which contain usually no erythrocytes but a
higher number of dead cells in comparison to fresh materials.
3. The ALDH staining should be performed in Aldefluor assay

buffer, which contained inhibitors of ATP-binding cassette
transporters and therefore prevent the efflux of Aldefluor fluo-
rescent dye from cells. The export of the dye is also inhibited
at low temperature, so that ALDH stained cells should be kept
in cold buffer after the staining.
4. Recap the tube and DEAB vial immediately because DEAB is
provided in 95 % ethanol.
5. The ALDH enzymatic reaction happens as soon as the Aldefluor
substrate is added to the cell suspension. DEAB, which is an
ALDH inhibitor, prevents the transformation of Aldefluor
substrate into its fluorescent product and should be mixed with
an aliquot of Aldefluor-stained cells without any delay.
6. The Aldefluor fluorescent substrate excites at 488 nm, and its
fluorescence is detected by using a standard FITC 530/30
band pass filter.
7. A density plot of FITC vs. SSC is recommended. It helps to
identify the ALDHbright cells (Fig. 1e).
8. The ALDH level of ALDHdim cells with high SSC is in general
slightly higher than those with low SSC, but less than that of
ALDHbright cells. Erythrocytes are negative for ALDH, lym-
phocytes display usually a lower level of ALDH compared to
myeloid cells, AML blasts and stem cells.
9. In a few patients, a high frequency of ALDHbright cells can be
observed, which is associated with their ability to develop AML
in xenografts. ALDH cannot be used for the separation of
HSC and LSC in these cases.
10. Cells can be sorted at a rate of about 5,000 cells per second.
It can be adjust depend on the sorting efficiency. Before sort-
ing in to FACS tubes with collection medium, vortex the tubes
to coat the surfaces with medium. The sort tube and collection
tubes have to be kept at 4 °C, as the Aldefluor dye can be
exported by the ABC transporter at a higher temperature.
After sorting, cells should be placed on ice to preserve their
viability.
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Chapter 20
Serial Orthotopic Transplantation of Epithelial Tumors

in Single-Cell Suspension
Heather A. McCauley and Géraldine Guasch
Abstract
Orthotopic transplantation of tumor tissue into recipient mice has long been established to study the
role of the microenvironment in tumorigenesis and metastasis. Many of these transplantation assays
involve the surgical implantation of an undissociated piece of tumor tissue. However, dissociation of
tumor tissue into a single cell suspension prior to orthotopic transplantation enables the injection of fewer
cell numbers, the selection of tumor-initiating populations by specific purification using antibody staining
and fluorescence-activated cell sorting, and the analysis of tumor-forming efficiency.
In this chapter, we provide a method to perform serial transplantation of tumor cells into their
niche of origin. Visualization of the location of transplanted tumor cells is essential to confirm the success
of the transplant as well as the viability of transplanted cells. We also describe an optimized immunofluo-
rescence protocol to visualize tumor cells shortly after transplantation. This serial transplantation protocol
allows for an experimental tumorigenesis assay to more closely mimic spontaneous tumor formation and is
applicable to many microenvironments.
Key words Niche, Microenvironment, Cancer, Tumor-initiating cells, Transplantation, Orthotopic
1 Introduction
The surrounding microenvironment, or niche [1], is an essential

component of tumorigenesis, tumor growth, and metastasis [2].
In some cases, such as in transition zones, two microenvironments
merge, which could contribute to tumor susceptibility [3].
Epithelial transition zones are defined by the junction between two
distinct types of epithelia [3] and may act as a stem cell niche [4–6].
They are often associated with the development of cancer in both
mice and humans. The distinctive microenvironment surrounding
transition zones may contain signaling cues from the two opposing
types of epithelia and this could contribute to cellular transforma-
tion. One such transition zone occurs at the junction between the
Keratin 14 positive (K14+) stratified squamous epithelia of the anal
231
232 Heather A. McCauley and Géraldine Guasch
canal and the simple epithelia of the rectum. This anorectal transition
zone is susceptible to squamous cell carcinoma (SCC) formation in
mice and humans associated with loss of TGFβ signaling [7–9].
Mice, in which the gene encoding the transforming growth factor-β
receptor II (TGFβRII) has been conditionally targeted in K14+
cells, develop spontaneous SCC in their anorectal region, while
TGFβRII deficient backskin appears morphologically normal [7].
Orthotopic transplantation is defined by the injection of
tumor cells or the transplantation of tumor tissue into anatomi-
cally appropriate sites, such as their microenvironment of origin.
This has the advantage of creating physiologically relevant pri-
mary tumors that can lead to spontaneous metastases in various
distant sites in contrast to induced metastasis formed after inject-
ing tumor cells in the blood circulation [10]. Placing cells into
their original niche improves their survival and proliferation, as
foreign microenvironments may not support tumor growth in the
same way and can cause misleading results [11]. Many of the pro-
tocols designed for surgical orthotopic transplantation involve
transplanting fragments of tumor, approximately 1 mm3, directly
into a recipient mouse without first dissociating the tumor into
a single-cell suspension [10]. However, dissociating a tumor into
a single-cell suspension prior to orthotopic transplantation is a criti-
cal step if the goal of the experiment is to identify a tumor-initiat-
ing population of cells [12].
Microenvironmental influence has been shown to affect cell
type differentiation. For example, hair follicle bulge stem cells
cultured on extracellular matrix from the corneal limbus and com-
bined with conditioned medium from limbal cells results in the
differentiation of those cells into corneal-like epithelium [13].
This microenvironmental reprogramming is also illustrated with
thymic epithelial cells that function as epidermal and hair follicle
stem cells when exposed to the microenvironment of the skin [14].
Therefore, orthotopic transplantation is crucial for the successful
outcome of tumorigenesis experiments if the goal is to create
tumors that mimic the original tumor.
A single-cell suspension of tumor cells can be sorted by flow
cytometry to dissociate distinct cell populations within a tumor.
This is accomplished by staining with fluorescent antibodies that
recognize cell surface proteins known to be expressed on cells that
display tumor initiating properties. Suspending cells in Matrigel, a
matrix of basement membrane proteins, ensures that the trans-
planted cells do not diffuse away from the surgical location and has
been shown to improve tumor formation [15].
Fluorescent labeling of tumor cells prior to orthotopic trans-
plantation is essential to control the accuracy of surgical technique,
to monitor the precise site of injection, and to visualize interactions
Serial Orthotopic Transplantation of Epithelial Tumors in Single-Cell Suspension 233
between tumor and microenvironment [10]. This can be

accomplished using retroviral infection of fluorescent proteins into
tumor cell lines [16, 17] or using a mouse reporter containing an
Enhanced Yellow Fluorescent Protein gene (EYFP) inserted into
the Gt(ROSA)26Sor locus [18] (Jackson Laboratory). Expression
of EYFP is blocked by an upstream loxP-flanked STOP sequence.
When bred to mice with a cre recombinase gene under the control
of a Keratin 14 promoter, the STOP sequence of the targeted gene
is deleted in epithelial tissue and EYFP expression is observed.
When mated with a mouse tumor model (TGFβRIIFlox/Flox × K14Cre
[7] × Rosa-Flox-Stop-Flox-EYFP in our case), the mouse reporter
will allow the lineage tracing of the cells of interest in vivo without
manipulating the cells in vitro. Moreover, this reporter marks the
lineage permanently, even if cells change properties and undergo
epithelial to mesenchymal transition as frequently seen in aggres-
sive tumors and during metastasis. Harvesting the tissue surround-
ing the transplantation site 3–5 days post-transplantation and
visualizing the fluorescent label is essential to ensure that the cells
localize to the proper niche. We have optimized the technique for
visualizing EYFP + SCC cells after transplant in the anorectal
transition zone.
This protocol begins with a tumor cell line of interest, fluores-
cently labeled. The first step involves transplanting these cells into
the microenvironment of origin in recipient mice, either immuno-
compromised or of a syngeneic background. Three to five days
after transplanting these cells, at least one mouse must be sacri-
ficed to visualize the location of the fluorescently labeled cells.
Depending on the aggressiveness of the tumor cells, as well as
the number of cells transplanted, tumors will begin to grow in the
recipient mice within 1–6 weeks. These tumors grafted from the
cell line can then be dissociated into a single cell suspension and
re-transplanted into the orthotopic microenvironment of a new
recipient mouse to test self-renewal properties of tumor cells.
Alternatively, antibody staining against cell surface markers
expressed on putative cancer stem cells and purification by
fluorescent-activated cell sorting can be employed at this step if
identifying a tumor-initiating population is of interest. This proto-
col ensures that an experimental tumorigenesis assay more closely
mimics the progression of a spontaneously arising tumor by keep-
ing the microenvironment consistent.
In this chapter, using our anorectal tumor model as an example,
we describe a novel protocol for orthotopic transplantation of ano-
rectal SCC cells into the anorectal transition zone. This protocol
is applicable to other transitional epithelial microenvironments,
such as the cervix (between the endocervix and ectocervix) or the
limbus (between the cornea and conjunctiva).
2 Materials
2.1 Cell Culture 1. Tumor cell line, fluorescently labeled (we use the
TGFβRII Flox/Flox ×K14Cre × Rosa-Flox-Stop-Flox-EYFP +
anorectal SCC cell line established in our laboratory).
2. Tissue culture dish, 100 × 20 mm (BD Falcon).
3. Epithelial cell culture medium with 0.05 mM Ca2+ [19] (add
83.5 μl of 0.05 M CaCl2 stock per 200 ml of medium).
4. Trypsin-EDTA, 0.25 % (Gibco).
5. Versene solution, sterile (dissolve 0.04 g EDTA disodium salt
(Sigma) in 20 ml 10× PBS) + 0.4 % glucose solution, sterile
(make a 25 % stock by dissolving 12.5 g glucose (Sigma) in
50 ml deionized water).
6. 1× Phosphate-buffered saline, sterile (1× PBS).
7. 15 ml conical tubes (BD Falcon).
8. Bright-Line Hemacytometer, 0.100 mm deep (Hausser
Scientific).
9. Trypan Blue Solution, 0.4 % (Sigma).
10. Centrifuge.
11. Sterile eppendorf tubes.
2.2 Surgery 1. Matrigel Matrix Basement Membrane, 5 ml vial (BD catalog

#356234).
2. Sterile barrier tips, p1000, p200 (Avant Guard).
3. Epithelial cell culture medium without calcium or serum [19].
4. 1 ml syringes with 27G × 1/2 needles (Becton Dickinson), kept
on ice.
5. Sterile cloth, tape, alcohol wipes, surgical gloves.
6. Class 100 HEPA filtered mass clean air portable table
(Biobubble, Fort Collins, CO).
7. Plastic drop-box (made internally by CCHMC plant
engineering).
8. Nose cone (made internally by CCHMC plant engineering).
9. Plastic hoses for anesthesia system (Butler Schein).
10. Isothesia isoflurane (Butler Schein).
11. Ohmeda anesthesia system with isoflurane vaporizer (Ohmeda
Isotec 4), with 100 % oxygen and delivering isoflurane at a
flow rate of 2.5 L/min.
2.3 Mice Female nude mice, approximately 6–8 weeks old (at the beginning
of the experiment), were used for these experiments. We maintain
a homozygous Nu/Nu strain in the laboratory, which can be
purchased from Charles River (see Note 1). Mice are housed in a
barrier facility using ventilated racks and automated watering that
provides a sterile environment for preventing infections. The air
supply for the barrier facility is class 100 HEPA filtered air. All
equipment, food and bedding are autoclaved prior to entry into
the barrier facility through a double door, floor loading autoclave.
Mouse cages are changed using a clean workstation and sterile for-
ceps. The forceps are sterilized, using a glass bead sterilizer,
between cages. Every month, a sentinel is evaluated for serology,
parasitology, and pathology. The barrier veterinary technicians
only work in clean areas and change into scrubs before donning a
sterile gown, mask, shoe covers, bonnet, and gloves. Passage
through an air shower is required to gain access to the barrier facil-
ity. The barrier facility is “clean,” with exception for Helicobacter
spp. and Murine Norovirus (MNV). It is negative for internal and
external parasites.
2.4 Tumor 1. Sterile Scissors, Iris Ribbon, 4″ (Sklar).

Dissociation 2. Sterile Dissecting Forceps, Half-curved 1 mm tip, 4″ (George
Tiemann & Co).
3. Sterile disposable scalpel, #21 (Sklar).
4. Tissue culture dishes, 100 × 20 mm and 60 × 15 mm (BD Falcon).
6. 1× Hanks’ Balanced Salt Solution (HBSS) (Gibco).
7. Fetal Bovine Serum (FBS), chelated [19].
8. Collagenase, from Clostridium histolyticum (Sigma). Prepare a
20 % stock by dissolving 1 g of powdered collagenase in 5 ml
1× sterile phosphate-buffered saline; aliquot 250 μl into eppen-
dorf tubes and store at −20 °C to avoid repeated freezing and
thawing that will decrease enzyme activity.
9. DNAse I, from bovine pancreas, 10 mg/ml (Sigma).
10. Rocking platform (VWR).
11. Precision gravity convection incubator, 37 °C (GCA
corporation).
12. 50 ml conical tubes (BD Falcon).
13. Sterile cell strainers, 70 and 40 μm (Fisher).
14. Trypsin-EDTA, 0.25 % (Gibco).
15. Epithelial cell culture medium without calcium [19].
16. Centrifuge.
2.5 Tumor Analysis 1. 4 % Paraformaldehyde solution, methanol-free (dilute 10 ml

16 % paraformaldehyde solution in 30 ml 1× phosphate-buffered
saline; keep at 4 °C) (Thermo Scientific).
3. 30 % sucrose (dissolve 30 g of sucrose (Sigma) in 100 ml of 1×

phosphate-buffered saline; keep refrigerated at 4 °C).
4. OCT Compound (Tissue-Tek).
5. 50 ml conical tube (Becton Dickinson).
6. Cryomold standard disposable vinyl specimen mold
(Tissue-Tek).
7. Dry ice.
8. Cryostat, HM550 series, Options O M V P D (ThermoFisher
Scientific).
9. Superfrost Plus microscope slides (Fisher).
10. Pap Pen (Research Products International Corporation).
11. Gelatin block (2.5 % Normal Goat Serum (Jackson
ImmunoResearch Laboratories), 2.5 % Normal Donkey Serum
(Jackson ImmunoResearch Laboratories), 1 % Bovine Serum
Albumin (Sigma), 2 % Gelatin (Sigma), 1 % Triton X-100
(10 %, Sigma) in 1× phosphate-buffered saline; aliquot 5 ml in
15 ml conical tubes and store at −20 °C).
12. Anti-green fluorescent protein antibody, conjugated to Alexa
Fluor 488, 1:1,000 dilution (Invitrogen).
13. 0.1 % Triton X-100 (Sigma) in 1× PBS.
14. Hoechst 33342, trihydrochloride, trihydrate, 1:2,000 dilution
(Invitrogen).
15. Antifade (dissolve 0.125 g p-phenylenediamine (Sigma) in
5 ml 10× phosphate-buffered saline; mix for 1 h and pH to
9.0; add 45 ml glycerol (Sigma) and mix well; store in 0.5 ml
aliquots at −80 °C and keep covered at all times, as antifade is
light sensitive).
16. Microscope glass (VWR).
17. Clear nail polish.
18. Zeiss Axio Imager motorized fluorescent microscope with a
high-resolution color Axiocam camera and software to generate
precise fluorescent images for localization of the tumor cells.
3 Methods
3.1 Preparation In vivo transplantation experiments assay the tumorigenicity of cancer

of Cells for cell lines while maintaining tumor-niche interactions. Established
Transplantation tumor cell lines, as well as primary tumor cells, can be used in ortho-
topic transplantation. Suspending cells in 30 % Matrigel before
transplantation improves engraftment by providing a matrix of
basement membrane proteins to which cells can attach. Keeping
Matrigel on ice at all times is imperative to the success of trans-
planting cells, as it becomes very viscous at room temperature.
3.1.1 Day 1 1. Thaw the 5 ml vial of Matrigel overnight on ice at 4 °C.

2. Cool all syringes, sterile pipette tips and eppendorf tubes that
will come in contact with Matrigel by placing at 4 °C
overnight.
3.1.2 Day 2 1. Aliquot 500 μl Matrigel in cold eppendorf tubes using a P1000
pipettor and cold tips. These 500 μl aliquots can be stored at
−20 °C until needed to avoid repeated thawing and freezing.
Prepare 30 % Matrigel by diluting Matrigel in cold epithelial
cell culture medium without calcium or serum. Calculate
150 μl 30 % Matrigel per injection. Keep 30 % Matrigel on ice
at all times.
2. Remove cells from the incubator and ensure that cells look
healthy and are approximately 80 % confluent.
3. Under a sterile hood, aspirate medium and wash cells with ster-
ile 1× PBS.
4. Combine equal volumes of 0.25 % Trypsin-EDTA and Versene
solution with 0.04 % glucose. Add 2 ml of this mixture to each
10 cm plate of cells. Incubate 5–8 min at 37 °C.
5. Vigorously pipette all around the plate with a P1000 pipettor
to detach cells and add to 5 ml epithelial cell culture medium
containing 0.05 mM of calcium in a 15 ml conical tube.
6. Centrifuge for 5 min at 450 rcf at room temperature.
7. Carefully aspirate supernatant. To avoid clumping of cells, gen-
tly tap the bottom of the 15 ml tube before adding 2 ml of
epithelial cell culture medium containing 0.05 mM calcium to
the pellet. Resuspend the cells by pipetting two to three times
with a 5 ml pipette to obtain a single cell suspension. Note that
this step is important to have an accurate count in the next step.
8. Using a hemacytometer and trypan blue solution, count the
number of viable cells in the suspension.
9. Transfer 100,000 viable cells per injection to an ice cold eppen-
dorf tube. Centrifuge cells in the eppendorf tube for 5 min at
450 rcf at 4 °C.
10. Carefully aspirate supernatant.
11. Resuspend cells in 150 μl 30 % Matrigel per injection using
a P200 pipettor and cold tips and keep on ice. If possible,
prepare enough cells with 30 % Matrigel for one more injec-
tion than planned to avoid loss of cells in the dead space of the
syringe used during the injection (see Note 2).
3.2 Transplantation Orthotopic transplantation of tumor cells into their microenviron-

of Cells into Their ment of origin allows for experimental tumorigenesis to more closely
Microenvironment mimic spontaneous tumorigenesis. As an example, anorectal squa-
of Origin mous cell carcinoma cells will be transplanted into the anorectal
transition zone of a recipient mouse (Fig. 1). All procedures are
Fig. 1 The surgical method of cell transplantation into the anorectal transition zone. (a) Surgical set-up within
a class 100 HEPA filtered mass clean air portable system (single asterisk) includes a surgical table (double
asterisk) and an anesthesia delivery system (triple asterisk) to minimize complications from transplantation.
(b) Mice can be placed into a drop-box receiving vaporized isoflurane and will fall asleep within 1–2 min.
(c) Once mice are asleep, they can be easily transferred to a nose cone delivering vaporized isoflurane to
remain unconscious. (d, d′) Once mice are anesthetized and placed into the nose cone, insert a cold, sterile
needle containing tumor cells suspended in 30 % Matrigel into the anorectal transition zone. Carefully move
the tip of the needle from side to side to create a space for the cells to reside (arrow). (e) After the cells are
dispensed, a swelling will appear in the injection site. A small bubble (white arrow) may escape from the
puncture wound, but this typically does not affect tumor formation (color figure online)
done under Institutional Animal Care and Use Committee (IACUC)

approval. For the transplantation experiments, all instruments must
be sterilized. All animals are observed daily by the animal care staff,
including weekends.
1. Anesthetize mice using isoflurane in a plastic drop-box and keep
them under anesthesia using a nose cone until the injection is
completed (see Note 3, Fig. 1a–c).
2. Draw cells into a cold syringe, ensuring that there are no bub-
bles. Remove bubbles by inverting the syringe and tapping to
let the air out.
3. Insert needle into the anorectal transition zone (see Note 4,
Fig. 1d, d′).
4. Before dispensing the cells, ensure that there is a space for the
cells to reside by carefully moving the tip of the needle from
side to side (Fig. 1d, d′).
5. Dispense 150 μl of cells in 30 % Matrigel. If the injection is
correctly done, no cells should come out from the injection
site (see Note 5, Fig. 1e).
6. Remove anesthetized mice from the nose cone and place in a
clean, sterile cage. Mice generally recover from isoflurane anes-
thesia within 1–2 min.
3.3 Analysis It is important to verify the location of cells after transplantation to

of Transplanted ensure the accuracy of transplantation technique as well as viability
Tumor Cells of cells. Using a cell line with a fluorescent label makes this verifica-
tion simple (Fig. 2). In order to amplify the signal of a weak fluo-
3.3.1 Verification
rescent tag, such as EYFP, immunofluorescent staining with an
of Location
antibody that recognizes both GFP and EYFP can be employed.
of Transplanted Cells
1. Three to five days after transplantation, sacrifice at least one
mouse by CO2 inhalation according to standard protocol, after
which death is determined by cessation of breathing and pinch-
ing the limbs. These procedures are consistent with the recom-
mendations of the Panel of Euthanasia of the American
Veterinary Medical Association.
2. Carefully dissect the region in which cells were transplanted.
3. Place tissue in a 50 ml conical tube in 10 ml 4 % paraformalde-
hyde overnight at 4 °C.
4. Decant the 4 % paraformaldehyde and wash tissue with 1× PBS
three times for 10 min.
5. Decant the PBS and place tissue in 10 ml 30 % sucrose over-
night at 4 °C. This step helps to preserve the morphology of
the tissue.
6. Decant the 30 % sucrose and replace with 10 ml fresh 30 %
sucrose + 5 ml OCT. Mix gently by inverting and place at 4 °C
overnight.
7. Embed tissue in OCT using a cryomold and dry ice. Store the
frozen block at −80 °C until ready to cut sections.
8. Cryosection tissue into 10 μm sections on Superfrost Plus slides.
It is important to use Plus slides to avoid loss of the fixed tissue
during the staining procedure, as Plus slides have a permanent
positive charge. Slides can be kept at −80 °C or directly stained.
9. Using a PAP pen, draw a hydrophobic barrier around tissue
sections.
10. Wash slide three times with 1× PBS.
11. Incubate slide in 0.1 % Triton (in 1× PBS) for 10 min to per-
meabilize tissue.
Fig. 2 Verification of location of transplanted EYFP + cells by immunofluorescent antibody staining. (a) Three to
5 days post-transplantation of tumor cells, the injected anorectal region of the mouse appears normal.
Dissection, fixation and staining of this tissue are required to verify the location of transplanted fluorescently
labeled cells. (b) EYFP + anorectal SCC cells injected into the anorectal region of a recipient mouse localize
precisely to the anorectal transition zone, as seen by immunofluorescent antibody staining (image taken using
a 10× objective lens). α6-integrin (red; BD Bioscience) stains the transplanted EYFP + cells. It is present in the
basal layer of the stratified epithelia of the anal canal. DAPI (blue) labels the nuclear chromatin
12. Block tissue with gelatin block for 1 h at room temperature.

13. Add Alexa-488-GFP antibody, diluted 1:1,000 in gelatin block,
to tissue (see Note 6). Incubate for 1 h at room temperature.
14. Wash slide in 0.1 % Triton three times for 10 min each.
15. Incubate tissue with Hoechst 33342 to stain the nuclei, diluted
1:2,000 in 1× PBS for 10 min at room temperature.
16. Wash once with 1× PBS.
17. Mount coverslip with antifade to preserve the fluorescence,
ensuring that there are no bubbles, and seal with nail polish.
Keep slides covered at all times, as the fluorescent antibody and
the antifade are both light sensitive.
18. Image on a fluorescent microscope to identify location of
EYFP + cells (Fig. 2b).
3.3.2 Harvesting, Depending on the aggressiveness of the tumor cell line, number of
Dissociation of Tumor, and cells injected and genetic background of recipient mice, tumor
Preparation of Tumor Cell latency can vary. 100,000 cells originating from an aggressive
Suspensions cancer cell line generally form a palpable and visible tumor in an
immunocompromised nude mouse by 1 week post-transplantation
(see Note 7, Fig. 3a).
1. Sacrifice tumor-bearing mice by CO2 inhalation according to
standard protocol, after which death is determined by cessa-
tion of breathing and pinching the limbs. These procedures are
consistent with the recommendations of the Panel of Euthanasia
of the American Veterinary Medical Association.
2. Carefully dissect the tumor, ensuring that it is separated from
skin, fat, muscle, and other contaminating tissue (Fig. 3b).
Remove any necrotic tissue.
Fig. 3 Harvesting, dissociation of tumor and preparation of tumor cell suspensions. (a) Between 1 and 6 weeks
post-transplantation, anorectal tumors will begin to grow. (b) Dissect the tumor, separating it from skin, fat and
other contaminating tissue and place it in a 10 cm plate containing 1× PBS. (c, c′) Large tumors (greater than
1 × 1 cm) can be separated into pieces that can be fixed and embedded for antibody staining as well as dis-
sociated for transplantation assays. (d) The piece of tumor must be minced into small pieces, approximately
1 mm in diameter. (e) After incubation with collagenase, the tumor mixture will appear very viscous. (f) After
treatment with DNAse I, the viscosity of the tumor mixture will be greatly reduced
3. If desired, slice the tumor into multiple pieces for embedding

and antibody staining. Our tumors grow to be approximately
2 × 2.5 cm at the time of sacrifice; only one quarter of this
tumor mass is dissociated according to this protocol (Fig. 3c, c′).
4. Place a small piece of tumor in 8 ml sterile 1× HBSS and mince
using a scalpel until all pieces are uniform and small (Fig. 3d).
This volume is optimized for a piece of tumor approximately
1 cm × 5 mm2.
5. Add 100 μl 20 % collagenase to dissociate the minced tumor.
Incubate for 45 min while shaking at 37 °C. We place a rocking
platform within a dry incubator set at 3 rpm. Note that collage-
nase activity decreases after many freezing-thawing cycles; there-
fore, store 20 % collagenase in 250 μl aliquots at −20 °C.
6. After 45 min, the tumor mixture should be homogeneous
and may appear viscous (Fig. 3e). Add 8.3 μl DNAse I to the
minced tumor and incubate for an additional 10 min while
shaking at 37 °C. After DNAse I treatment, the viscosity of the
tumor mixture should be greatly reduced (Fig. 3f).
7. Using a 25 ml pipette, add 10 ml cold 1× PBS to the minced
tumor and pipette vigorously eight to ten times to further
dissociate the tumor mixture. Add the minced tumor to a
50 ml conical tube.
8. Wash the plate twice with cold 1× PBS to obtain the maximum
number of cells extracted from the tumor and add to the same
50 ml conical tube.
9. Centrifuge for 10 min at 200 rcf at 4 °C.

10. Aspirate the supernatant. Using a 10 ml pipette, resuspend the
cell pellet with 20 ml cold 1× PBS and add 200 μl chelexed FBS.
11. Place a 70 μm filter atop a new 50 ml conical tube. Pre-wet the
filter with 1× PBS and filter the tumor cells.
12. Place the 70 μm filter in a 60 mm culture dish and add 2 ml
0.25 % Trypsin-EDTA to the filter to maximize the number of
cells extracted from the minced tumor. Incubate for 5 min at
37 °C.
13. After 5 min, block the activity of the trypsin by adding 5 ml
epithelial cell culture medium to the filter. Add the filtered
trypsinized cells to the filtered cells in the same 50 ml conical
tube from step 10.
14. Place a 40 μm filter atop a new 50 ml conical tube and pre-wet
with epithelial cell culture medium. Using a 5 ml pipette, pass
the cells through the 40 μm filter.
15. Centrifuge for 10 min at 200 rcf at 4 °C.
16. Aspirate the supernatant and resuspend the cell pellet in epi-
thelial cell culture medium without serum to create a single-
cell suspension (see Note 8).
3.4 Transplantation 1. Count cells dissociated in Subheading 3.3.2 using a hemocy-

of Dissociated Tumor tometer and trypan blue as in Subheading 3.1, step 8.
into Microenvironment 2. Transfer 100,000 viable cells to a cold eppendorf tube
of Origin (see Note 2).
3. Centrifuge cells in the eppendorf tube for 5 min at 200 rcf at
4 °C.
4. Carefully aspirate supernatant with a pipette, ensuring that the
cell pellet remains intact.
5. Resuspend cells in 150 μl 30 % Matrigel and keep on ice. Avoid
forming bubbles while resuspending.
6. Transplant into the original microenvironmental niche, as
described in Subheading 3.2.
4 Notes
1. The strain and the age of mice used for transplantation assays
can affect the results of tumorigenic assays. Using immuno-
compromised mice may result in faster and more aggressive
tumor formation, but can produce more artificial results than
transplantation into immunocompetent (syngeneic) mice.
The age of the nude mice may influence the reproducibility of
the graft. To keep this parameter consistent, we maintain our
own nude colony by mating heterozygous Nu+/Nu females,
which are not immunodeficient and are haired, with homozy-

gous male Nu/Nu to obtain Nu/Nu offspring. This ensures
that age-matched mice will be used for each experiment.
2. Resuspending cells in 150 μl 30 % Matrigel per injection
ensures that there is sufficient volume within the syringe for a
successful transplant. For multiple transplants of the same cells,
simply resuspend the desired number of cells in 150 μl 30 %
Matrigel per injection. We suggest calculating sufficient cells
and 30 % Matrigel volume for at least one more injection than
planned to account for the dead space in the syringe.
3. We found the use of inhaled isoflurane more convenient than
intraperitoneally injected anesthetic for many reasons. Firstly,
this avoids an unnecessary intraperitoneal injection. Secondly,
mice recover faster (within 1–2 min), thus avoiding the use of
a warm bed. Thirdly, our mice have never died as a result of
inhaled anesthesia in contrast to mice given intraperitoneally
injected anesthetic.
4. The anorectal transition zone occurs between the stratified
squamous epithelia of the anal canal and the simple epithelia
of the rectum, approximately 3 mm from the external opening of
the anus in mice [3].
5. A small bubble of Matrigel may escape from the puncture site
(Fig. 1e). Also, the junction between the syringe and needle
creates a “dead volume” that will slightly reduce the amount of
Matrigel injected. While these events may slightly decrease the
total number of cells transplanted, the loss is not significant
enough to affect tumor formation in our experience.
6. Costain with other tissue markers if desired. If using an anti-
body that requires secondary staining, Alexa-488-GFP can be
left on the slides overnight at 4 °C.
7. Alternatively, tumors that arise spontaneously in genetically
modified mice [7, 20] may be dissociated and transplanted
according to this protocol without placing cells in culture.
However, depending on the aggressiveness and proliferative
potential of these cells, secondary tumors arising from fresh
primary tumors may not grow as quickly or grow as consis-
tently as tumors created from an established cell line.
8. Dissociation of the tumor into a single-cell suspension is nec-
essary for a successful orthotopic transplant using Matrigel.
However, the tumor-forming efficiency of these bulk tumor
cells can be improved by sorting these cells by flow cytometry
based on cell surface marker expression to enrich for a tumor-
initiating cell population. If sorting, resuspend the cell pellet
in 2 % chelated fetal bovine serum and stain cells with anti-
bodies according to manufacturer’s guidelines. The use of
chelated serum avoids clumping of cells. Collect sorted cell
populations in 500 μl epithelial cell culture medium without

serum in eppendorf tubes. The sorted cells can then be centri-
fuged for 5 min at 200 rcf at 4 °C and resuspended in 150 μl
30 % Matrigel per injection.
Acknowledgments
This work was supported by the Concern Foundation and part by

PHS Grant P30 DK 078392. G.G. is a Sidney Kimmel Scholar and
a V Foundation Scholar.
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Chapter 21
Isolation and Propagation of Colon Cancer Stem Cells

Pramudita R. Prasetyanti, Cheryl Zimberlin, Felipe De Sousa E. Melo,
and Jan Paul Medema
Abstract
The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native
environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a
detailed methodology for the isolation and expansion of both human colon CSCs and mouse intestinal
adenoma together with a brief differentiation and coculture method that proved to be valuable to study
the concept of CSCs plasticity.
Key words Colorectal cancer, Adenoma, Cancer stem cells, CD133, Wnt signaling, Ultralow adherent,
EGF, Isolation
1 Introduction
During the last decade, the hierarchical organization that is present

within malignancies has been clearly attributed to a stem-cell-like
subset of tumor cells. These so-called cancer stem cells (CSCs)
have gained an exclusive interest as they are believed to fuel tumor
growth [1, 4], seed distant metastasis [2], and are responsible for
therapy failure and tumor resistance [1, 6].
In colon cancer, a CSC population has been identified based
on the expression of immature cell surface markers, such as CD133
[4, 5], or high activity of the canonical Wnt signaling pathway [8].
Predictably, many efforts have been made to design therapies that
will specifically eradicate the CSC pool [1, 6, 8]. An essential step
to succeed in this endeavor is the establishment of sophisticated
culture methods that faithfully maintain tumor stem cell features in
tissue culture, from both human and transgenic mouse material.
Here we describe a method to isolate and culture human primary
colon CSCs and mouse adenomas. The tumor pieces are enzymati-
cally digested and cultured in serum free modified neurobasal
First two authors contributed equally.
247
248 Pramudita R. Prasetyanti et al.
medium supplemented with defined growth factors. This method

of culture has proven to be superior to the classical establishment
of colorectal cancer cell lines that requires selective clonal out-
growth of tumor cells after primary cultures enter culture crisis
[3, 6]. Colon CSCs cultures maintain self-renewal capacity as well
as the potential to differentiate into all lineages present in the origi-
nal malignancy in vitro and in vivo [1, 8]. The molecular mecha-
nisms that drive and maintain the CSC population have started to
emerge and the identification and origin of factors that maintain or
even induce a CSC phenotype remains an intense area of research.
We have recently shown that colon CSCs features can be installed
even in more differentiated tumor cells by stromal cells present in
the intestinal microenvironment [1, 7]. We provide here a con-
cise method to study the effect of stromal derived factors on the
stem cell and differentiation potential of colon CSCs. Despite
their usefulness human colon CSCs have one major drawback
and that is that can not be transplanted into an immune-competent
host and thus cannot be used to study interactions between can-
cer and the immune system. To this end we routinely use cultures
derived from adenomas and carcinomas originating from geneti-
cally modified mice. A method to culture these adenoma cells is
provided as well.
2 Materials
2.1 General 1. Laminar flow cabinet certified for handling biosafety level 2
Equipment and (BSL-2) specimens.
Material List 2. Humidified tissue culture incubator capable for maintaining
37 °C and 5 % CO2 atmosphere.
3. Low-speed centrifuge (e.g., Hettich Rotanta 460).
4. Inverted and phase contrast light microscopes equipped with
10×, 20×, and 40× magnification.
5. Hemocytometer counting chamber.
6. 37 °C waterbath.
7. Trypan blue solution 0.4 % (Sigma).
8. Pipet-aid.
9. Vortex.
10. Sterile disposables plastics: 100 μl filter tips, 1,000 μl long
reach pipette tips (VWR), pipettes 1-, 2-, 10-, and 25-ml
volume, 15 and 50 ml polypropylene sterile tubes (Falcon),
10 cm petri dish.
11. Cell culture vessels: 24-, 6-wells plate ultralow adherent
plate (Corning), 25 cm2 ultralow adherent flask (Corning),
24-, 12-, 6-wells adherent plate (Greiner), 25-, 75-cm2 adherent
plate (Greiner).
Isolation and Propagation of Colon Cancer Stem Cells 249
Table 1
Overview of reagents for CSC medium and isolation enzymes
Component Supplier Stock solution Working dilution Storage (°C)

Culture medium
Advanced DMEM/F12 Invitrogen 4
N2 supplement Invitrogen 100× 1 in 100 −20
Glucose 45 % Sigma 2.5 M 3.4 in 1,000 4
Trace element B VWR 1× 1 in 1,000 4
Trace element C VWR 1× 1 in 1,000 4
Hepes 1 M Invitrogen 5 mM in dH2O 1 in 100 4
Heparine Sigma 2 mg/ml in dH2O 1 in 1,000 4
Insuline Sigma 10 mg/ml 1 in 1,000 4
Beta-Mercaptoethanol Sigma 50 mM in dH2O 1 in 500 4
L-glutamine Lonza 200 mM 1 in 500 −20
Growth factors
Human EGF Peprotech 20 μg/ml in DMEM/F12 1 in 1,000 −20
Human b-FGF Tebu-Bio 50 μg/ml in DMEM/F12 1 in 5,000 −20
Digestion enzyme
Collagenase type IV Sigma 100 mg/ml in PBS 1 in 100 −20
Hyaluronidase type IV-S Sigma 10 mg/ml in PBS 1 in 500 −20
2.2 Tissue Digestion 1. Equipment: closed container certified to transport BSL-2 speci-
of Human men, medical waste container, sharps waste disposal container.
Tumor Tissue 2. Autoclaved dissection equipment (thumb dressing forceps,
dissecting scissors straight 10 cm, scalpel handle, and disposable
blade number 22).
3. Sterile disposable plastics: 1.8 ml cryovial tube (Nunc), 70 μM
nylon mesh filter (Nunc).
4. Hank’s Balanced Salt Solution 1× (HBSS) without Ca2+ and
Mg2+ (Invitrogen).
5. Antibiotic and antimycotic solution: 100 U/ml penicillin,
100 μg/ml streptomycin, 10 mg/ml gentamycin, and 2.5 μg/ml
amphotericin-B (see Note 1).
6. Culture medium, digestion enzymes and growth factor (see
Table 1 and Note 2).
7. Digestion buffer: digestion enzymes (see Table 1) in culture
medium (see Note 3).
8. Ficoll gradient reagents (e.g., Lympholyte—Miltenyi)
2.3 Culture and 1. Phosphate-Buffered Saline (PBS) without Ca2+ and Mg2+.
Differentiation of 2. Trypsin 10× solution (Lonza). To prepare working solution,
Isolated Human CSCs dilute the trypsin 1: 40 in PBS. Store at 4 °C.
Table 2
Overview of reagents for Adenoma culture medium
Working
Component Supplier Stock solution dilution Storage
Adenoma culture medium
Advanced DEMEN/F12 (ADF) medium Invitrogen 4 °C
B27 supplement Invitrogen 50× 1 in 50 −20 °C
N2 supplement Invitrogen 100× 1 in 100 −20 °C
BSA 20 % 1 in 50 4 °C
GlutaMAX-I Invitrogen 100× (200 mM) 1 in 100 RTa
Hepes Invitrogen 1M 1 in 100 4 °C
N-acetyl cysteine Sigma-Aldrich 500 mM 1 in 500 4 °C
Antimycotic/Antibiotic Invitrogen 100× 1 in 100 −20 °C
Mouse EGF Prepro-Tech 1 μg/ml 1 in 20 −20 °C
Mouse noggin Prospec 10 μg/ml 1 in 100 −20 °C
a
RT room temperature
3. Trypsin inhibitor 1× solution (Sigma). To prepare working

solution, dilute 3.3 ml of trypsin inhibitor in 500 ml of PBS.
Store at 4 °C.
4. Non-heat-inactivated Fetal Bovine Serum Gold (FBS-Gold)
(PAA). Store at −20 °C.
5. Differentiation medium: culture medium supplemented with 2 %
of FBS-Gold.
6. Colonic Myofibroblast cell line 18Co (ATCC, CRL-1459)
cultured in DMEM medium supplemented with 10 % FCS
and 1 % glutamine.
2.4 Reagents for 1. Sterile surgical tools for dissection (scissors, tweezers, round-
Mouse Adenoma tipped curved oral gavage for rats).
Isolation and 2. Fresh Intestinal adenomas derived from mice.
Passaging
3. Sterile disposable plastics: 10 ml syringe, 50 and 15 ml poly-
propylene sterile tubes (Falcon), 15 cm tissue culture dishes,
24-well tissue culture plate, 70 μm nylon mesh filter.
4. Phosphate-Buffered Saline (PBS) without Ca2+ and Mg2+.
5. 2.5 % 10× trypsin, no phenol red (Invitrogen catalog #
15090-046).
6. Advanced DMEM/F12 (ADF) medium (Invitrogen catalog #
12634-010) (see Note 4).
7. Growth Factor Reduced Matrigel (BD Bioscience, catalog #
354230) (see Note 5).
8. See Table 2 for adenoma culture medium.
3 Methods
All human tissue specimens should be assumed infectious and

therefore all appropriate BSL-2 practices should be followed.
Aseptic techniques should be exercised in all steps of the isolation
and propagation of colon CSCs. All the methods mentioned below
are performed at room temperature unless indicated otherwise.
3.1 Isolation 1. After surgical resection, immediately transfer the tumor piece
of Colon CSCs into sterile 50 ml tubes containing HBSS. As indicated in
BSL-2 practice, use appropriate container to transport the
specimen to the laminar flow cabinet (see Note 6).
2. Place the tumor piece in a new 50 ml tube containing 20 ml of
HBSS and incubate at room temprature for fifteen minutes.
Shake the tube vigorously to wash the remaining feces and
blood from the tumor pieces. Allow the tumor piece to sedi-
ment and discard the supernatant. Repeat this step twice.
3. Transfer the tumor piece in a 5 cm petri dish containing 5 ml
of HBSS. Using a sterile blade, cut the tumor piece into two
parts and remove as much necrotic tissue as possible.
4. Transfer the tumor fragments into a new 50 ml tube containing
20 ml of HBSS. Wash the tumor fragment (see points
Subheading 3.1, step 2) and repeat this step a minimum of ten
times or until the supernatant is clear.
5. Cut the tumor fragment into small pieces of approximately
0.5 mm3 (see Note 7).
6. Transfer the minced tumor into a 50 ml tube containing diges-
tion buffer. Digest the tumor for 30–60 min at 37 °C with
occasional agitation (see Note 8).
7. Allow the digested fragments to sediment to the bottom of
the tube. Transfer the supernatant, which now contains the
dissociated single cells, into a new 50 ml tube and centrifuge at
600 × g for 5 min. The supernatant can be discarded or used for
a second round of digestion (see Note 9).
8. Resuspend and pass the supernatant through a 70 μm nylon
mesh filter. Pass additional 10 ml of culture medium to wash
the remaining cells away.
9. Centrifuge the cells suspension at 600 × g for 5 min.
10. Discard the supernatant and resuspend the cell pellet with
10 ml of culture medium and centrifuge the cells again at
600 × g for 5 min. Repeat this step to remove any trace of
digestion enzymes.
11. Resuspend the pellet in a minimal volume of pre-warmed culture
medium (usually 1 ml) supplied with growth factor and aliquot
10 μl for cell counting.
Fig. 1 Phase contrast micrographs of CSCs derived from stage IIIA colon carcinoma. (a) Enzymatically dissoci-
ated tumor are seeded as suspension culture in ultralow adherent plate (10× magnification) (b) Substantial
increase of dead cells are occasionally observed during the first days of plating (10× magnification) (c) After
30 days, the CSCs will proliferate faster than in the beginning of culture initiation. The solid healthy spheres
appear as balls of cells that can be maintained over passages (40× magnification)
12. Determine the number and viability of cells using hemocy-

tometer counting chamber with trypan blue exclusion
(see Note 10).
13. Seed 5–10 × 104 cells/cm2 in ultralow adherent plate or flasks
containing culture medium supplemented with growth factors.
14. A large amount of dead cells usually appear in the first days of
culture initiation. During this period, colon CSCs cultures
should initiate small spheroid structures (Fig. 1). To maintain a
viable culture, it is recommended to change the culture medium
every 4 days and passage the cells according to the following
procedure (see Subheading 3.2).
3.2 Culture and 1. Examine the confluency and condition of the cells under in the
Propagation of Colon microscope prior to passaging (see Note 11).
CSCs 2. Transfer the cell suspension into a 15 ml tube. Wash the culture
vessel with 2 ml of PBS to detach the remaining cells and pool
them together in the previous tube.
3. Centrifuge the cells at 450 × g for 3 min and discard the
supernatant.
4. Resuspend the pellet in trypsin and incubate at 37 °C for 5 min
(see Note 12).
5. Stop the trypsin reaction by adding similar volume of trypsin
inhibitor and dissociate the spheres mechanically by pipetting
up and down several times using a long reach pipette tip.
6. Add 5 ml of culture medium on top of the cells suspension and
centrifuge at 500 × g for 3 min. Gently decant the supernatant
without disturbing the pellet.
7. Resuspend the pellet in culture medium supplemented with
growth factor in the desired cell density (see Subheading 3.1,
steps 11–13).
Fig. 2 The withdrawal of growth factor and the addition of serum induce CSCs differentiation. After 96 h of
incubation with the differentiation medium, the CSCs sphere structure will completely disappear into a flattened
morphology. This can be observed in a monolayer culture system (a) and in Matrigel (b) (40× magnification)
3.3 Differentiation The conditions described above maintain colon CSCs in an immature
of Colon CSCs undifferentiated state. However, withdrawal of the growth factors
and addition of serum can lead to the differentiation of colon CSCs
into the various differentiated lineages present in the original
malignancy. This differentiation process can be performed at least
in two different ways: in adherent conditions as a monolayer cul-
ture (Fig. 2a) and in a 3D Matrigel culture system (Fig. 2b).
Conversely, the addition of myofibroblasts derived factors prevents
the differentiation potential of colon CSCs (Fig. 3) [7].
3.3.1 CSCs 1. Dissociate the spheres according to the Subheading 3.2 and
Differentiation in Adherent count the cells with the hemocytometer counting chamber.
Cell Culture Vessel 2. Transfer the cells into a 15 ml tube and pellet the cells by cen-
trifugation 600 × g for 3 min.
3. Resuspend the cells in 5 ml of PBS to wash the residual growth
factors. Spin the cells at 500 × g for 3 min and discard the
supernatant. Perform this washing step twice.
4. Resuspend the pellet using p1000 tips in pre-warmed culture
medium containing EGF and seed the cells according to the
recommended density in adherent culture vessels.
Fig. 3 Myofibroblast conditioned medium induce dedifferentiation. Serum-induced morphological differentiation

is prevented in the presence of MFCM derived from the colonic cell line 18Co (40× magnification)
5. After 24 h, aspirate the culture medium and gently wash the

attached cells with PBS. Perform this washing step twice.
6. Overlay the pre-warmed differentiation medium on top of
the cells.
7. Observe the morphology of the cells after 48 h.
3.3.2 CSCs 1. Follow the steps 1–3 described in Subheading 3.3.1.

Differentiation in Matrigel 2. Resuspend the cell pellet in pre-warmed differentiation
medium into approximately 6 × 105 cells/ml.
3. Mix 50 μl of Matrigel with 50 μl of cell suspension (see Note 13).
4. Directly transfer the mixture into 1 well of a pre-warmed
24-well-adherent plate and incubate at 37 °C for 15 min until
the Matrigel suspension solidifies.
5. Gently overlay 1 ml of differentiation medium on top of the
Matrigel.
6. Observe the morphology of the cells after 48 h.
3.3.3 Generation of 1. Seed 1 × 106 18Co human myofibroblasts cells in a T75 adherent
Myofibroblasts Conditioned Flask in DMEM medium supplemented with 10 % FCS (fetal
Medium (MFCM) calf serum) and 1 % glutamine.
2. The following day, wash the cells two times with PBS to remove
any remaining FCS.
3. Delicately overlay 10 ml of DMEM/F12 on the myofibroblasts
(see Note 14).
4. Collect the myofibroblasts conditioned medium (MFCM)
after 24 h in a 15 ml falcon.
5. Spin the medium collected at 750 × g for 5 min (see Note 15).
6. MFCM can be aliquoted and stored at 4 °C up to 6 months
(see Note 16).
3.3.4 Dedifferentiation 1. Follow the steps 1–3 of CSCs differentiation in adherent cell
of Colon-CSCs with MFCM culture vessel as described in (see Subheading 3.3.1).
2. Resuspend the pellet using p1000 tips with MFCM supple-
mented with 2 % heat inactivated FBS (see Note 17).
3. Observe the morphological changes after 48 h (see Fig. 3c).
3.3.5 Dedifferentiation of 1. Seed 7.5 × 105 18Co human myofibroblasts in a T25 flask in
Human Colon-CSCs on a DMEM culture medium supplemented with 10 % FCS and
Feeder Layer of 1 % glutamine.
Myofibroblasts 2. When cells are approximately 90 % confluent, aspirate the
medium and wash the cells twice with PBS.
3. Overlay 4 ml of differentiation medium on the myofibroblasts.
4. Follow steps 1–3 of CSCs differentiation in adherent cell
culture vessel (see Subheading 3.3.1) and resuspend 1 × 105
of CSCs with 1 ml of differentiation medium.
5. Transfer the CSCs suspension onto the T25 flask containing
myofibroblasts and swirl gently.
6. Observe the morphological changes after 48 h.
3.4 Mouse Adenoma Unless otherwise stated all steps are carried out at room temperature
Isolation and do not need to be done in sterile conditions.
1. Carefully remove the small intestine from the mouse (see
Note 18) and place it on a clean flat surface (see Note 19).
2. Flush the intestine (see Note 20) with 4 °C PBS to remove the
remaining feces.
3. Cut the intestine longitudinally open with a sterile scissors
(see Note 21) and open up the intestine so that the villi face
upwards.
4. Cut out adenomas using a scissors (see Note 22) and dice the
tissue into small pieces (approximately 0.3 × 0.3 mm).
5. Place adenoma pieces into a 50 ml tube containing 10 ml 4 °C
PBS. From this step onwards the adenoma pieces should be
kept on ice.
6. Wash the adenoma pieces three to five times with 10 ml 4 °C
PBS (see Note 23) or until the supernatant is clear.
7. Remove supernatant and add 5 ml 2.5 % 10× trypsin. Incubate
for 30 min at 37 °C.
8. Shake vigorously and collect the supernatant in a new 50 ml
tube. Keep the supernatant on ice for the remaining steps.
9. Repeat washing steps of the adenoma pieces two to four times
using 10 ml 4 °C ADF medium (see Note 24). After each wash
collect the supernatant in the same 50 ml Falcon tube.
10. Filter the collected supernatant through a 70 μM mesh filter.
Fig. 4 The isolation and expansion of Adenoma cultures. The isolation of adenomas can be done by isolating
lesions which are visible either macroscopically (a) or microscopically (b-5× magnification). The adenomas
are already large by day 10 (c) and can be maintained indefinitely (40× magnification)
11. Centrifuge the filtered supernatant at 600 × g for 5 min.

12. Remove supernatant and wash the adenoma cell pellet one to
two times with 10 ml 4 °C ADF medium (see Note 25).
13. Conduct the remaining steps under sterile conditions.
14. The adenoma cells are counted and in each well of a pre-heated
24 well plate (see Note 26) plate approximately 500 cells resus-
pended in 50 μl Matrigel (see Note 27). Subsequently, medium
supplemented with all growth factors is added on top.
15. After 1–3 days the cells start forming rounded spheres (see
Fig. 4). Refresh the medium every 3–4 days and passage them
on a weekly basis (see Note 28).
3.5 Passaging All steps are done in sterile conditions.

of Adenomas
1. Remove medium from the well.
2. Pre-wet pipette tip (p1000) (see Note 29) and disrupt the
spheroids in Matrigel by pipetting up and down.
3. Add 2 ml ADF media into a 15 ml falcon tube and pipette
the disrupted Matrigel structures from step 2 into this tube
(see Note 30).
4. Centrifuge at 600 × g for 3 min (see Note 31).
5. Remove as much medium as possible and resuspend the cells
in 50 μl Matrigel (see Note 28 ). The split ratio for the
adenoma cells is normally 1:4.
4 Notes
1. The microbic flora naturally present in the colon is a major

source of contamination during the establishment of colon
CSCs cultures. Therefore, antibiotics and antimycotic solution
need to be supplemented in HBSS, used during the isolation
procedure.
2. Pre-warm the culture medium to 37 °C in a water bath. Place

the warmed medium at room temperature for 30 min after
incubation in the water bath.
3. Pre-warm the digestion buffer at 37 °C in the water bath
15 min prior to the digestion step.
4. For the washing steps in the adenoma isolation procedure,
ADF medium is used without any additional factors.
5. To shorten thawing time, we aliquot the Matrigel in 1 ml.
Matrigel is thawed on ice at 4 °C overnight or it can be placed
on ice approximately 1 h before use.
6. Isolate the CSCs as soon as possible once the tumor has been
resected from the patient. Digesting the tumor pieces within
a few hours after the resection will increase the chance of estab-
lishing a viable culture.
7. The size of the tumor fragments crucially impact the release of
single cells during the digestion step. We recommend placing
the tumor fragments into a cryovial tube containing 500 μl of
digestion buffer and subsequently cut it vigorously with
scissors. This method successfully reduces the size of the tumor
fragments.
8. Use 10 ml of digestion buffer for every 1 cm3 of tumor piece.
Maintain the tube containing tumor and digestion mix 37 °C
in a water bath. Shake the tube vigorously every 5 min to
release the single cells from the bulk of the tumor.
9. It is extremely important to avoid over digestion of the tumor
pieces. Approximately 1 cm3 of tumor piece can be optimally
digested for 30–40 min with this protocol. However, every
10 min the digestion mixture should be examined under the
microscope to look for clumps of dead cells as a sign of over
digestion. If this sign appears but there are still large pieces of
tumor left to digest, then proceed with the digestion in two
steps. In this case, one can obtain the first fraction of single
cells by continuing the isolation process as mentioned in
Subheading 3.1, step 8. Subsequently, use the supernatant
that still contains active enzymes to digest the tumor piece left
to obtain the second fraction of single cells.
10. Occasionally it is impossible to remove all the red blood cells
and the necrotic part of tumor. When the fraction of red blood
cells and/or necrotic cells represents more than 70 % of the
viable tumor cells, we recommend performing a ficoll gradient
step to purify epithelial cells according to the manufacturer’s
protocol.
11. We have observed that the density of spheres influence the
proliferation of colon CSCs. Spheres should be passaged when
individual spheres contains 100–500 cells.
12. We recommend using 500 μl of trypsin for 1–5 × 106 cells.

To avoid over digestion, examine the cells under the micro-
scope after 5 min. If big spheres are still observed, extend the
trypsin incubation until the spheres are evenly dissociated.
13. Matrigel clogs quickly inside the tips and can cause inaccurate
measurement as well as introduce bubbles in the Matrigel-cell
suspension. To avoid this problem, keep the Matrigel on ice
during culturing and resuspend the cells and Matrigel as quick
as possible.
14. The medium used to derive MFCM is the culture medium
without growth factors.
15. Alternatively, MFCM can be filtered through a 0.4 μm nylon
mesh filter.
16. MFCM is used at 1:2 dilution in culture medium containing
growth factors.
17. For dedifferentiation, use MFCM with 2 % FBS without any
additional factors.
18. The easiest way to remove the intestine is to cut the junction
between the stomach and the esophagus. Grab the stomach
with forceps, gently lift and pull, as the stomach rises cut away
the tissue attached to the intestine, without damaging the
intestine. In this manner both the intestine and colon should
untangle, making them easy to remove.
19. We find it easiest to use round 15 cm tissue culture dishes,
as they are large enough to easily accommodate the mouse
intestine and colon.
20. For the flushing, the intestine/colon needs to be rinsed inside
and out with 4 °C PBS. We use a round-tipped curved oral
gavage for rats attached to a 10 ml syringe to flush the intestine.
The stomach and ceacum first need to be removed, by cutting at
the attachment sites with the duodenum and colon respectively.
The opening of the intestine or colon is then placed over the
entry point of the oral gavage tube and slowly the intestine is
filled with PBS. It is important not to fill the intestine to quickly
but rather allow the PBS to pass at a passive rate. By lifting the
small intestine with the oral gavage, gravity can help the PBS to
pass at a passive rate.
21. By segmenting the intestine into three sections, the intestine is
easier to cut open longitudinally.
22. Depending on the size of the adenomas, they can either be
visualized by macroscopically or microscopically, after which
the adenomas then can be removed from the intestine.
23. The washing is performed in order to remove remaining debris.
When washing, shake the 50 ml tube vigorously for 5 seconds,
allow adenoma pieces to settle and discard supernatant. Repeat
this procedure by adding 10 ml 4 °C PBS.
24. The washing is done as described in Note 23, but using 10 ml

4 °C ADF instead of 10 ml 4 °C PBS
25. For a large pellet, two washing steps are necessary in order to
remove all traces of trypsin. However, if the pellet is small wash
once to avoid excessive cell loss.
26. The 24-well plate is pre-heated for at least 1 h at 37 °C. This helps
the solidification of the Matrigel upon plating.
27. For suspension in Matrigel the cells need to be spun down
(3 min, 600 × g) and the supernatant removed. Using 50 μl of
Matrigel per well, resuspend the cells and drop the Matrigel in
the center of the well. The plate is then placed at 37 °C for
10 min after which the 500 μl of medium per well is added.
Avoid making bubbles when resuspending the cells, as this can
affect the visualization of structures in the Matrigel.
28. The frequency of passaging is dependent on the rate of adenoma
growth. Optimally, they should be passaged just before they
become overly large, dense or filled with debris.
29. We pre-wet the pipette tip with medium. It is important to do
before sucking up the Matrigel, as it might stick to the inside
of the tip.
30. It is important to mechanically disrupt the structures as much
as possible by pipetting up and down at least ten times.
31. After centrifugation the cells can be seen at the bottom of the
tube. A separate transparent layer of Matrigel can be seen
above the cells. The Matrigel can easily be aspired and removed.
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INDEX
A cellular and noncellular elements ..................................25

confocal microscope......................................................31
Aberrant markers (AM) description ....................................................................25
candidates ...................................................................221 dissection methods
CD34 expression ................................................225, 226 adult ovaries ............................................................29
lymphoid lineage markers ...........................................219 fixation ....................................................................30
Acid phosphatase (ACP) staining larval ovaries .....................................................29–30
components ........................................................105–106 Drosophila niches ..........................................................26
HLPSC, calcium phosphate surface ...................108, 109 glycerol and vortex ........................................................31
spherolites ...................................................................116 goat serum ....................................................................30
ACP staining. See Acid phosphatase (ACP) staining immunostaining protocol..............................................32
Acute myeloid leukemia (AML) incubation and washing steps .......................................32
and ALDH (see Aldehyde dehydrogenase (ALDH)) larval ovary and adult germaria ...............................25–26
and AM materials
candidates .............................................................221 antibodies..........................................................28–29
CD34 expression ..........................................225, 226 DNA staining and mounting ..................................29
lymphoid lineage markers .....................................219 fixation and washing ...............................................28
and HSC (see Hematopoietic stem cells (HSCs)) fly husbandry ..........................................................27
and LSC (see Leukemia stem cells (LSC)) laser scanning confocal microscope.........................29
materials ovary dissection .......................................................27
and antibodies .......................................................220 “peanut butter-like”.......................................................30
flow cytometry ......................................................221 Adult SVZ. See Subependymal zone (SEZ)
gradient separation, MNC ....................................220 AFS. See Amniotic fluid stem (AFS) cells
patient samples .....................................................220 Aldehyde dehydrogenase (ALDH)
methods data acquisition and gating strategies .................223, 224
ALDH staining ....................................................222 description ..................................................................219
data analysis ..................................................225–226 erythrocyte lysis ..................................................221–222
erythrocyte lysis ............................................221–222 FISH analysis .............................................................227
instrument setting and data acquisition ........223–225 high activity (ALDHbright)...................................219, 224
isolation, MNC.....................................................221 HSC and LSC separation ..........................................226
patient samples .....................................................221 instrument settings .....................................................226
sorting, HSC and LSC .................................226–227 lower level (ALDHdim) .......................................219, 224
staining, surface markers ...............................222–223 metabolic processes .....................................................219
trypan blue ..................................................................227 vs. SSC plot ................................................................225
Adenoma staining .......................................................................222
mouse adenoma isolation ....................................255–256 Alkaline phosphatase (ALP) staining
passaging ....................................................................256 aliquots .......................................................................108
reagents and culture medium ......................................250 components ................................................................105
Adult stem cells diazocoupling technique .....................................108, 109
antibody staining ..........................................................30 HLPSC cells and surrounding sockets .......................116
cell-cell interactions ................................................25–26 and OC.......................................................................115
DOI 10.1007/978-1-62703-508-8, © Springer Science+Business Media, LLC 2013
261
STEM CELL NICHE: METHODS AND PROTOCOLS
262 Index
ALU Primer Mix..............................................................209 HSC populations ..........................................................50

AML. See Acute myeloid leukemia (AML) isolation .................................................. 70–71, 122–124
Amniotic fluid stem (AFS) cells MSCs extraction.....................................................67–68
adherent cells ..............................................................196 niche, DTCs (see Disseminated tumor cells (DTCs))
antibiotics ...................................................................196 screen
CD117 expression ...................................... 191, 192, 196 application ..............................................................79
cell expansion......................................................195, 196 BMMNC ...............................................................81
components, immune-selection ..................................193 cell processing/apheresis laboratories,
coverslips ....................................................................196 clinical centers.............................................79–80
culture medium and plastic ware ........................192–193 procure ....................................................................81
cytograms ...........................................................196, 197 as stromal cells ..............................................................44
flow cytometry analysis ...............................................193 Stro-1 positive fraction .................................................68
methods, immune-selection ........................................195 transplantation ..............................................................43
morphology ................................................................196 Bone marrow stromal cells (BMSCs) .................................68
performance, samples..................................................196 Boudreaux, M. ..................................................................131
procedure ....................................................................191 Brain
purification, homologous population ..........................192 freezing procedure and slices collection ......................144
sample seeding ............................................................194 osmotic minipump (see Osmotic minipump
Animal model, DTC. See Disseminated tumor cells (DTCs) system, brain)
Artificial osteogenic niches
ALP and OC secretion...............................................115 C
artificial matrices ........................................................104 Calcium phosphate (CP) surface
cell suspension ............................................................115 ACP stained HLPSC .........................................109, 110
CP coatings (see Calcium phosphate (CP) surface) composite plates .........................................................104
ectopic osteogenesis test .............................................104 OC stained HLPSC ...................................................110
heat-treated copper sulfate crystals .............................114 SEM image
HLPSC strain FL-42 .........................................113–114 fibroblast-like HLPSC .........................................112
HSC ...........................................................................103 interconnected sockets ..........................................117
materials osteoblast-like HLPSC ........................................113
ACP staining ................................................105–106 rounded HLPSC ..................................................112
ALP staining ........................................................105 spherolites with pores ...........................................117
cultural and fixative components ..........................105 Cancer, orthotopic transplantation. See Orthotopic
immunocyto-chemical components ......................106 transplantation, epithelial tumor cells
light microscopy....................................................106 Cancer stem cells (CSCs)
samples .................................................................104 colorectal cancer (see Colorectal cancer, CSCs)
SEM .............................................................106–107 description ..................................................................247
methods Cannula
cells fixation ..........................................................108 brain infusion kit ........................................................137
diazocoupling technique ...............................108, 109 and catheter ................................................................138
immunocytochemical detection ....................109–110 location .......................................................................138
reflecting light microscopy ............................110–111 penetration, LV...........................................................136
SEM ..................................................... 111, 112, 113 and skull .....................................................................139
short-term cultivation ...................................107–108 Cap cells (CpCs)
MMSC .......................................................................104 L3 larvae .......................................................................26
nuclei counterstain, hematoxylin/azure.......................115 subset of antibodies.......................................................28
regression curve, ALP-stained sites ............................116 Cardiomyocytes
SEM image.................................................................117 ECM ..........................................................................153
supplemented osteogenous medium ...........................114 embryogenesis ............................................................154
AutoMACS buffer ................................................. 84, 95, 99 magnification ..............................................................158
Cardiovascular diseases .....................................................153
B
CD133..............................................................................247
Bone marrow (BM) CD146
and BMSCs ..................................................................68 expression
ECM extracts ...............................................................44 cells viability .....................................................94–95
ficoll ..............................................................................49 CXCL12 and Angiopoietin1 ..................................92
Index
263
flow-cytometry .......................................................92 gelatin removal......................................................157
primary and sorting stromal cells ......................92, 93 hyperglycemia .......................................................160
functional hematopoietic ME.......................................78 LIF inhibition ...............................................157, 160
stromal cells based ........................................................82 MMP9 expression ........................................158, 159
CD117 expression ............................................ 191, 192, 196 Pasteur pipette ......................................................157
Cell cycle regions ..................................................................158
human myogenic ........................................................166 TIMP4 expression ........................................158–159
phase distribution .......................................................174 transformation ......................................................157
progression..................................................................166 GSCs ..............................................................................1
quiescence, human myogenic cells ..............................172 Disseminated tumor cells (DTCs)
synchronization, human myogenic cells......................170 dormancy ....................................................................207
Chazaud, B. .............................................................. 166, 167 HSCs ..................................................................207–208
c-Kit, AFS cells. See Amniotic fluid stem (AFS) cells materials
Collagen matrix IHC, detection .....................................................210
3D scaffolds ..................................................................52 isolation, FACS.....................................................209
polymerization reagents ................................................46 PCR, detection .....................................................209
Sirius Red assays ...........................................................46 methods
Colorectal cancer, CSCs detection, IHC .............................................212–213
culture methods ..................................................247–248 detection, PCR .............................................210–211
materials isolation, FACS.............................................211–212
adenoma culture medium .....................................250 PCa, xenograft model .................................................208
culture and differentiation ............................249–250 DMD. See Duchenne muscular dystrophy (DMD)
equipment and material list ..................................248 Donor satellite cells
medium and isolation enzymes .............................249 function ......................................................................183
tissue digestion, tumor tissue ................................249 grafting .......................................................................183
methods preparation..................................................................183
adherent cell culture vessel, pre-treatment
differentiation .........................................253–254 grafting ................................................. 186, 188–189
culture and propagation ........................................252 test function ..........................................................186
isolation ........................................................251–252 pure population, mouse...............................................185
Matrigel, differentiation .......................................254 Dormancy .........................................................................207
MFCM .........................................................254–255 Drosophila
mouse adenoma isolation ..............................255–256 adult SC (see Adult stem cells)
myofibroblasts, differentiation ..............................255 ovaries, GSCs
passaging, adenomas .............................................256 AP and HRP ........................................................1–2
self-renewal capacity ...................................................248 dissecting and staining ..........................................3–4
CpCs. See Cap cells (CpCs) and Dpp....................................................................1
Cystoblast (CB) ..................................................................13 DSHB ......................................................................6
Cyst progenitor cells (CPCs) fatten flies .................................................................3
anterior apex, testis .......................................................10 fixative buffer ............................................................5
and GSCs (see Germline stem cells (GSCs)) fluorescence-labeled adult ovaries .........................5, 6
hub cells ..................................................................10, 11 GSCs ........................................................................1
MARCM system ..........................................................14 immuno-label proteins..........................................1–2
materials ...............................................................2–3
D microfuge tube ..........................................................6
Developmental Studies Hybridoma Bank (DSHB) mounting and presentation ...................................4–5
anti-Hts ..........................................................................6 multiple structures and signaling molecules ..........1–2
anti-Lamin C .................................................................4 optimum number, flies ..............................................6
To-pro3 .......................................................................... 5 ovariole .....................................................................7
Dexter, T.M. ........................................................... 76, 89, 90 SC identification (see Stem cells (SCs))
Differentiation DSHB. See Developmental Studies Hybridoma Bank
donor mouse models ................................... 182, 184, 189 (DSHB)
ESC DTCs. See Disseminated tumor cells (DTCs)
and EBs ........................................................158, 160 Duchenne muscular dystrophy (DMD) ...........................180
264 Index
E materials and antibodies .......................................220

Fluorescence-activated cell sorting (FACS)
Ebbe, S. ............................................................................131 AML
ECM. See Extracellular matrix (ECM) ALDH staining ....................................................222
Embryo, HSCs. See Hematopoietic stem cells (HSCs) FACScan analyzer and FACSAriaII sorter...........223
Embryonic stem cells (ESC) HSC and LSC sorting ..........................................226
differentiation .............................................................153 immunophenotyping ............................................221
and EB........................................................................161 snap-cap tubes ......................................................220
and ECM ...................................................................153 Aria sorter.....................................................................99
frozen tubes ................................................................160 AutoMACS buffer .......................................................84
gelatin removal ...................................................160–161 CD146-based sorting, MSCs .................................93, 95
hemocytometer ...........................................................161 DTC isolation ............................................ 209, 211–212
homogenous cell suspension .......................................161 Follicle stem cells (FSCs)
LIF, mouse ..................................................................154 germarium ....................................................................13
materials niche and behavior ........................................................13
components...........................................................155 progenitor cells .............................................................13
culture medium components, mouse.............154–155 type, stem cell ...............................................................12
differentiation .......................................................155 Franklin, K.B. ...................................................................149
MEF inactivation .................................................155
medium.......................................................................161 G
methods
differentiation (see Differentiation) Germarium
ESC culture ..................................................156–157 antibodies .....................................................................28
MEF culture .........................................................155 larval ovary and adult ..............................................26–27
and MMP9 .........................................................153–154 ovariole .........................................................................26
non-irradiated MEFs .................................................160 Germline stem cells (GSCs)
therapy ........................................................................153 and CB .........................................................................13
and TIMP4.................................................................154 clones, mutant...............................................................17
transplantation ............................................................153 and CPCs .....................................................................10
Enhanced Yellow Fluorescent Protein (EYFP) gene Drosophila ovaries
cell culture ..................................................................234 antibodies..................................................................6
expression ...................................................................233 and cystblast .............................................................5
verification of location ........................................239–240 differentiation ...........................................................1
Epidermal growth factor (EGF)....................... 249, 250, 253 heterologous somatic cells.........................................1
ESC. See Embryonic stem cells (ESC) non-cell autonomous fashion ....................................1
Extracellular matrix (ECM) fertility and fecundity ...................................................10
HSC microenvironment ...............................................44 gametes production.......................................................10
laminin/fibronectin .......................................................44 and GB .........................................................................10
MMP9........................................................................153 germarium ....................................................................12
MMSC .......................................................................104 and gonialblasts ............................................................10
viscoelastic connective tissue .........................................44 testes and ovaries ..........................................................10
EYFP. See Enhanced Yellow Fluorescent Protein GFRA1
(EYFP) gene and GDNF ...................................................................36
IHC protocols ..............................................................42
F immunostaining ............................................................36
SSC characterization ....................................................36
FACS. See Fluorescence-activated cell sorting (FACS)
Glial cell line-derived neurotrophic factor (GDNF) ..........36
Fetal cells
Godt, D. .............................................................................28
Bovine Serum (FBS) .................................. 192, 209, 235
Gonialblast (GB) ................................................................10
FCS ............................................................................254
GSCs. See Germline stem cells (GSCs)
Flow cytometry
DTC isolation ............................................................209
H
HSC and LSC
advantages.............................................................218 Hematopoietic niche (HN)
immunophenotyping ............................................221 and BM ........................................................................43
instrument setting .................................................223 collagenase digestions/PBS...........................................54
Index
265
2D and 3D coculture system ...................... 45–46, 50–52 methods ........................................................169, 170
and ECM .....................................................................44 Pax7 expression ...........................................................175
ficolling .........................................................................54 Hypoxia
fluorochrome-labeled primary antibodies .....................45 FoxO genes .................................................................200
harvesting cells 3D coculture ............................46, 52–54 HIF and molecular action...................................201, 202
heparin..........................................................................45 materials
LSK cells ......................................................................50 antibodies..............................................................201
and OB .........................................................................44 chemicals ..............................................................201
osteoblast preparation .............................................44–48 methods
positive displacement pipette ........................................54 Ab staining ...........................................................203
stain and wash cells .......................................................50 IHC ......................................................................203
stromal cells preparation ...................................44, 48–49 stem cell antigens ..................................................202
Hematopoietic stem cells (HSCs) MMP degradation ......................................................201
AML neurogenic niches .......................................................200
and ALDH (see Aldehyde dehydrogenase (ALDH)) Nox .............................................................................200
in ALDH-AML samples ..............................226–227 regenerative medicine .................................................200
surface markers .....................................................219 ROS production .........................................................200
CD31 and CD34..........................................................58 Hypoxia-inducible factor-1 (HIF-1)
collagenase ....................................................................63 antibodies ...................................................................201
extraembryonic mesoderm ............................................57 description ..................................................................201
and HN (see Hematopoietic niche (HN)) detection .....................................................................203
and IHC .................................................................58–61 molecular action..................................................201, 202
laser capture microdissection ...................... 59–60, 62–63
marker expression .........................................................58 I
and MMSC ................................................................104
Image J
self-maintenance.........................................................103
angle tool.......................................................................40
stored slides ..................................................................63
“ctrl + M”.......................................................................40
treatment time ..............................................................64
download and open.......................................................38
YS and P-Sp/AGM................................................57–58
optimal resolution .........................................................38
HIF-1. See Hypoxia-inducible factor-1 (HIF-1)
paintbrush tool ..............................................................39
HLPSC. See Prenatal stromal cells of human lung (HLPSC)
seminiferous tubule .......................................................39
HN. See Hematopoietic niche (HN)
software ........................................................................38
Horseradish peroxidase (HRP)
Immunohistochemistry (IHC)
antibody-tagged enzyme.............................................1–2
detection, DTCs ......................................... 210, 212–213
light-emitting product ....................................................2
and HSC ................................................................58–61
HSC. See Hematopoietic stem cells (HSCs)
hypoxia ...............................................................203, 204
Human
OC detection .............................................. 106, 109–110
RCs (see Human RCs)
Immunostaining
stem cells, AFS (see Amniotic fluid stem (AFS) cells)
adult SC (see Adult stem cells)
Human leukocyte antigens (HLA)
GSC (see Germline stem cells (GSCs))
and DTCs
multiple structures and signaling molecules....................2
and FACS .............................................................208
Isolation
FITC anti-human HLA-A,B,C
colon CSCs.........................................................251–252
Antibody .................................................209, 212
mouse adenoma ..................................................255–256
visualized HLA-A,B,C positive cells....................213
HLA-DR PE and HLA-ABC FITC ................193, 197 L
Human RCs
isolation Laser capture microdissection (LCM)
material .................................................................166 anti-c-Kit antibody .......................................................63
methods ........................................................168–169 diethyl pyrocarbonate .............................................59–60
myogenic cell quiescence embed placentas and slice tissues ..................................62
material .........................................................166–167 fluorescent c-Kit-positive cell .......................................63
methods ........................................................169–171 microcentrifuge tubes ...................................................63
myogenic cell synchronization Lasko, P...............................................................................28
material .........................................................166–167 Laufer, E.............................................................................28
266 Index
Leukemia inhibitory factor (LIF) FSChighSSChigh “cells” ..........................................131–132

culture medium components ......................................154 GFP/RFP mice ..................................................126, 129
ESC culture medium ..................................................155 and HSC ....................................................................121
Leukemia stem cells (LSC) immunofluorescence image ................................121, 122
and ALDH (see Aldehyde dehydrogenase (ALDH)) immunohistofluorescence analysis ..............................132
in ALDH-AML samples ....................................226–227 materials
CD34+ CD38-cell population .....................................218 bone marrow .........................................................122
surface markers ...........................................................219 fluorescence activated cell sorting .........................123
Light microscopy .............................................. 106, 110–111 immunomagnetic cell separation ..................122–123
Lineage-Sca1+ ckit+ (LSK) cells methods
calculation.....................................................................51 bone marrow .................................................123–124
flow cytometric cell .......................................................50 fluorescence activated cell sorting .................125–126
markers .........................................................................50 immuno magnetic cell separation .................124–125
Long-term culture (LTC) nozzle sizes, flowcore cytometry facility .....................129
Dexter cultures..............................................................90 Nuclear Red ................................................................130
Hs27a and Hs5 .............................................................82 ploidies .......................................................................131
maintenance..................................................................90 post-sorting ................................................................131
medium formulations ...................................................83 purity ..................................................................130–131
MSC (see Marrow stromal cells (MSCs)) targeting problems ......................................................132
primary bone marrow ...................................................76 tibia, femur and iliac crest ...........................................129
LSC. See Leukemia stem cells (LSC) unwanted hemopoietic cells ........................................130
LSK cells. See Lineage-Sca1+ ckit+ (LSK) cells McKearin, D.......................................................................28
LTC. See Long-term culture (LTC) Mesenchymal stem cells, Stro-1.
Lymphocyte separation medium (LSM) See Stro-1 immunoselected MSCs
maintenance..................................................................73 MFCM. See Myofibroblasts Conditioned Medium
red blood cells removal .................................................71 (MFCM)
Microenvironment (ME)
M cultures ...................................................................78–79
Magnetic-activated cell sorting (MACS) CXCL12, SCF and ANGPT1 .....................................76
AutoMACS ...................................................... 84, 95, 99 definition ................................................................77–78
and flow sorters.............................................................82 epithelial transition zones ...........................................231
preparation, buffer ........................................................70 fluorescently labeled....................................................233
Stro-1 isolation .......................................................71–72 hematopoietic ...............................................................76
Magnetic selection, AFS ..........................................193, 195 heterozygote SL/SLd mouse ........................................75
Marrow stromal cells (MSCs) and HN ........................................................................43
and LTC and HSC ......................................................................44
cultures ...................................................................89 in vivo............................................................................ 76
growth ....................................................................82 influence .....................................................................232
primary (see Primary MSCs) mesenchymal origin ......................................................76
Matrix metalloproteinase-9 (MMP9) misleading misnomer ..............................................76–77
activation ....................................................................158 niches........................................................................9–10
differentiation .....................................................158, 159 orthotopic transplantation ..........................................232
function ..............................................................153–155 reprogramming ...........................................................232
regulation ............................................................158, 160 stroma/stromal cells ......................................................76
Mature megakaryocytes (MM) transplantation
B-cells and myeloid cells.....................................129–130 of cells ...........................................................237–239
BM cells .....................................................................122 of dissociated tumor..............................................242
CD41 expression ........................................................131 orthotopic (see Orthotopic transplantation,
cell lysis, NH4Cl ........................................................130 epithelial tumor cells)
centrifuge antibodies...................................................129 MicroRNA (miRNA)...................................................96–98
crushing and enzyme treatment ..................................132 Minipump. See Osmotic minipump system, brain
debris and cell clumps.................................................129 MMP9. See Matrix metalloproteinase-9 (MMP9)
DNA replication .........................................................121 MMSC. See Multipotent mesenchymal stromal cells
“enrich” mode .............................................................131 (MMSC)
FITC-CD41 ..............................................................130 Morphology, seminiferous epithelium ................................37
Index
267
Morphometry, SSCs. See Spermatogonial stem cells (SSCs) microenvironments ...................................................9
Mosaic analysis with a repressible cell marker well-defined structures............................................10
(MARCM) .................................................14, 17 in vertebrates (see Spermatogonial stem cells (SSCs))
Mouse Nocodazole synchronization
embryonic fibroblast (MEF).......................................155 arrested cells................................................................174
RCs (see Murine RCs) concentrations.............................................................174
Multipotency ....................................................................200 DMSO ...............................................................173, 174
Multipotent mesenchymal stromal cells (MMSC) human myogenic cell
and ECM ...................................................................104 material .................................................................167
osteogenic markers .....................................................104 methods ........................................................169, 170
treatment ....................................................................104 mutagen ......................................................................174
Murine RCs
coat petri dishes ..........................................................171 O
EDL and soleus muscles.............................................171 OB. See Osteoblasts (OB)
FACS-sorted satellite cells..........................................171 OC. See Osteocalcin (OC)
isolation Orthotopic transplantation, epithelial tumor cells
conical centrifuge tube and transfer definition ....................................................................232
collected cells ..................................................173 EYFP (see Enhanced Yellow Fluorescent
differentiation medium .........................................173 Protein gene (EYFP))
growth medium ....................................................173 fluorescent labeling .............................................232–233
materials .............................................................167–168 materials
matrigel-coated culture dishes ....................................173 cell culture.............................................................234
molecular analysis .......................................................173 mice ..............................................................234–235
multinucleated myotubes ............................................176 surgery ..................................................................234
myotubes.....................................................................173 tumor analysis ...............................................235–236
trypsinate ....................................................................173 tumor dissociation ................................................235
Muscle regeneration, skeletal muscle methods
analysis harvesting, dissociation and preparation .......240–242
donor-derived fibers .............................................184 microenvironment of origin ..........................237–238
donor-derived satellite cells ..................................184 preparation of cells ........................................236–237
muscle freezing ...........................................................184 transplantation of dissociated tumor .....................242
process ................................................................179–180 verification of location ..................................239–240
Myofibroblasts Conditioned Medium (MFCM) microenvironmental influence ....................................232
dedifferentiation, colon-CSCs ....................................254 Osmotic minipump system, brain
generation ...................................................................254 adult MF1 mouse .......................................................139
Myogenesis .......................................................................165 cell and gene therapy ..................................................136
Myogenic progenitor cells. See Human RCs; Murine RCs coordination, mouse brain ..........................................136
injection and infusion .................................................136
N
location .......................................................................136
Neural progenitor cells, SEZ. See Subependymal zone (SEZ) materials
Neural stem cell niche ......................................................135 brain infusion kit ..................................................137
Niches chemicals and reagents..........................................137
adult SC (see Adult stem cells) instruments ...........................................................137
CD31 and CD34..........................................................58 surgery ..................................................................137
c-Kit-positive HSCs .....................................................58 methods
description ..................................................................231 preparation....................................................137–138
Drosophila ovaries (see Drosophila) surgery ..........................................................138–139
epithelial transition zones ...........................................231 neural stem cell niches ................................................135
in vivo transplantation ................................................236 skull protection ...........................................................135
LCM (see Laser capture microdissection (LCM)) Osteoblasts (OB)
orthotopic transplantation (see Orthotopic C57Bl/6 pups ...............................................................48
transplantation, epithelial tumor cells) cultured stromal cells ....................................................50
proliferation and differentiation....................................58 endosteal region ............................................................44
SC identification flow cytometry ..............................................................48
GSCs/CPCs ...........................................................10 precursor cells ...............................................................47
268 Index
Osteocalcin (OC) bovine serum albumin .............................................83

concentration, aliquots ................................................108 cell and tissue culture ..............................................79
immunocyto-chemical components ............ 106, 109–110 citrate saline solution ........................................84–85
Ovary DMEM ..................................................................83
and adult germarium ....................................................26 EDTA.....................................................................84
dissection ......................................................................27 fetal bovine serum and horse serum ........................83
immunostaining and microscopy ................ 15–16, 18–19 ficoll solution ..........................................................85
and isolation of testis ..............................................14, 18 flow-cytometry and sorting ....................................82
Oxygen hemolytic buffer......................................................85
description ..................................................................199 human stromal cell lines .........................................82
diffusion distance ........................................................200 IRB approval and personnel training ......................79
hypoxia (see Hypoxia) and LTC growth medium .......................................82
partial pressure ............................................................199 LTC medium ..........................................................83
PBS-EDTA ............................................................84
P reverse transfection reagents ...................................85
Pasut, A. ...........................................................................168 solutions and buffers ...............................................84
Paxinos, G. .......................................................................149 stromal cell line medium .........................................83
Pimonidazole............................................................201, 203 trypsin-EDTA ........................................................85
Ploidy ME (see Microenvironment (ME))
megakaryocytes...........................................................121 methods
MM based .......................................... 126, 127, 128, 131 BMMNC .........................................................87–89
Nuclear Red ................................................................130 CD45 and CD14 ....................................................95
populations .........................................................130, 132 CD146high expression ........................................92–95
Polymerase chain reaction (PCR) .............................210–211 Ficoll density gradient separation .....................87–89
Prenatal stromal cells of human lung (HLPSC) hemolysis, bone marrow washout ...........................87
and ACP .....................................................................109 hMSC cultures .................................................91–92
and ALP .............................................................111, 116 long-term cultures ............................................89–90
3D-culture and establishment ............................112, 118 marrow screens .................................................86–87
fibroblast-like cells ...................................... 112, 113, 114 siRNA/ miRNA ...............................................96–98
FL-42 .........................................................................113 stromal cell lines .....................................................96
and OC.......................................................................110 Proliferation, SEZ
osteoblast ....................................................................113 anatomical references, mouse and rat..................146, 149
plastic surface..............................................................109 anterior-posterior and dorsal-ventral axes ..........146, 148
rounded ......................................................................112 BrdU preparation................................................142–143
suspension, osteogenic medium ..................................107 data processing....................................................147, 149
Pre-treatment, skeletal muscle division .......................................................................152
donor satellite cells (see Donor satellite cells) microscope and software .............................................146
host muscle tissue random selection.........................................................146
cryoinjury ..............................................................183 sampling methodology ...............................................146
grafting, donor-derived nuclei ......................186–188 statistical analysis ........................................................149
irradiation .....................................................181–182 Prostate cancer (PCa)
myotoxin injection ........................................182–183 HSC niche..................................................................208
Primary MSCs xenograft model ..........................................................208
aliquots .........................................................................98
R
biological characteristics .........................................97–98
CFU-Fs ........................................................................98 Regenerative medicine......................................................200
complete/near-complete hemolysis...............................99 Reserve cells (RCs)
FACS Aria sorter..........................................................99 growth and regeneration .............................................165
horse serum...................................................................99 human RCs (see Human RCs)
Hs27a and Hs5 .............................................................99 identification...............................................................174
isolation ........................................................................97 isolation, murine .................................................167–168
materials murine (see Murine RCs)
antibodies................................................................85 primary myogenic cell cultures ...................................166
AutoMACS/FACS buffer ......................................84 replated .......................................................................174
bone marrow screens .........................................79–81 self-renewal and myogenic differentiation ..................165
Index
269
Reverse-transfection Skeletal stem cells (SSCs)...................................................67
reagents.........................................................................85 Skull
stromal cell line, siRNA/ miRNA ..........................96–98 and cannula.................................................................139
lateral ventricles ..........................................................138
S lateroventral and rostro-caudal characteristics ............136
Sacchetti, B. ........................................................................92 osmotic minipump......................................................137
Satellite cells protection ...................................................................135
donor (see Donor satellite cells) surface .........................................................................139
location, niches ...........................................................179 Small interfering RNA (siRNA) ..................................96–98
skeletal muscle (see Skeletal muscle) Spermatogonial stem cells (SSCs)
Scanning electron microscopy (SEM) 5-bromo-2′-deoxyuridine (BrdU).................................41
adherent cells, glutaric dialdehyde solution.................108 components/cells ..........................................................35
CP coating irregularity ...............................................117 cytoarchitecture ............................................................36
and HLPSC (see Prenatal stromal cells fixation and embedding ................................................41
of human lung (HLPSC)) germ cell association .....................................................42
rough calcium phosphate surface ................................117 materials
Schofield, R. ............................................................... 44, 103 necessary materials ..................................................38
SC identification. See Stem cells (SCs) preserved/fixed and embedded specimens ..............36
Self-renewal. See Muscle regeneration, skeletal muscle spermatogonial morphology/phenotype ...........36, 37
SEZ. See Subependymal zone (SEZ) method
Shen, Y. ............................................................................126 data analysis ............................................................41
Short-term culture, CP-coated titanium distribution analysis ..........................................38–41
specimens ................................................107–108 image acquisition ....................................................38
siRNA. See Small interfering RNA (siRNA) microenvironment ........................................................35
Skeletal muscle morphological/phenotypical characterization ...............36
adult............................................................................179 testis..............................................................................35
anesthesia....................................................................189 Stem cells (SCs)
blue nuclei ..................................................................189 ESC (see Embryonic stem cells (ESC))
differentiation .............................................................174 identification
DMD .........................................................................180 asymmetric division ................................................13
donor mouse strain .............................................181, 182 cell-based therapies ...................................................9
donor satellite cell (see Donor satellite cells) culturing Drosophila .......................................... 13–14
dytrophin production ..................................................189 differentiated cells .....................................................9
host mouse strain ........................................................181 Drosophila testis (see Drosophila)
mdx mouse models......................................................180 embryos ............................................................16, 19
myoblasts ....................................................................175 fast red working solution ........................................21
myogenic cell FSCs .......................................................................13
cultures .................................................................165 generation, germline clones ....................................17
differentiation .......................................................166 germline and somatic clones ...................................14
growth and proliferation .......................................174 GSCs (see Germline stem cells (GSCs))
myotube preparation ...................................................174 in situ hybridization ........................ 12, 16–17, 19–21
nocodazole ..........................................................173–174 immunofluorescence staining,
Pax7 expression ...................................................174, 175 testis and ovary ...........................................18–19
pre-modulation, recipient mouse muscles immunostaining and microscopy ......................15–16
cryoinjury ..............................................................185 isolation, testis and ovary ..................................14, 18
irradiation .............................................................184 RNase .....................................................................21
myotoxin injury.....................................................185 somatic clones ...................................................17–18
surgical procedure .................................................185 under-and over-proliferation ....................................9
Pyronin Y fluorescence ...............................................175 muscle (see Skeletal muscle)
radiation dose .............................................................189 niche
RCs (see Reserve cells (RCs)) HN (see Hematopoietic niche (HN))
regenerated muscle fibers (see Muscle regeneration, hypoxia (see Hypoxia)
skeletal muscle) quiescent .....................................................................179
satellite cells ................................................................165 skeletal muscle (see Skeletal muscle)
trypsination.........................................................174, 176 Stereology .........................................................................142
270 Index
Stereotaxic frame ......................................................137, 138 proliferation assessment, BrdU preparation ........142–143

Stro-1 immunoselected MSCs protection ...................................................................150
BMSCs .........................................................................68 stereology....................................................................142
bone marrow aspirates ............................................67–68 streptavidin .................................................................151
cell suspension, pipetting ..............................................72 temperatures ...............................................................149
iPS cells ........................................................................67 Substances administration, osmotic minipump.
LSM .............................................................................73 See Osmotic minipump system, brain
materials
alpha-MEM medium .............................................69 T
blocking buffer ........................................................69 Testis
hybridoma supernatant .....................................69–70 anti-β-galactosidase ......................................................11
MACS buffer..........................................................70 components/cells ..........................................................35
methods cytoarchitecture ............................................................36
bone marrow isolation ......................................70–71 immunofluorescence staining .................................18–19
lymphocyte separation medium ..............................71 immunostaining and microscopy ............................15–16
MACS Stro-1 isolation ....................................71–72 morphophysiology ........................................................42
multipotent stem cells ...................................................67 and ovary isolation ............................................14, 18–19
positive nuclear staining and DAPI ..............................68 Tissue inhibitor of metalloproteinase-4
Stro-1 expression ..........................................................68 (TIMP4) ......................................... 154, 158, 159
Stromal cells Topography, SEZ .............................................................141
BM cells .......................................................................54 Transplantation
cytokines and growth factors ........................................44 ESC ............................................................................153
Subependymal zone (SEZ) orthotopic (See Orthotopic transplantation,
antibody ......................................................................151 epithelial tumor cells)
avoid freezing and thawing .........................................150 Tumor-initiating cells .......................................................232
corpus callosum ..........................................................150
estimation, proliferation......................................141–142 U
estimations..................................................................151
external horseradish peroxidase (HPR) ......................151 Ultralow adherent .....................................................248, 252
fluorescence immuno-histochemistry .................151–152
V
hematoxylin ................................................................151
immuno-histochemistry Visualization
antibodies..............................................................143 proliferation and differentiation....................................58
solutions................................................................143 P-Sp/AGM region and placenta ..................................58
isopentane ...................................................................150 Volk, T. ...............................................................................28
methods
brain freezing procedure and slices collection .......144 W
BrdU injection ......................................................144
Watson, C.........................................................................149
immuno-histochemistry,
Wnt signaling .............................................................76, 247
BrdU and Ki67 .......................................144–146
proliferation assessment ................................146–149 Z
orientation, brain ........................................................150
position .......................................................................142 Zammit, P.S. .....................................................................168

Stem Cell Niche

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Methods in

Molecular Biology 1035

Kursad Turksen Editor

For further volumes:

Methods and Protocols

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Library of Congress Control Number: 2013941996

© Springer Science+Business Media, LLC 2013

Printed on acid-free paper

Humana Press is a brand of Springer

Toronto, ON, Canada Kursad Turksen

1 Immunostaining of Germline Stem Cells and the Niche

Shen Y. Heazlewood, Brenda Williams, Melonie J. Storan,

RANA ABOU-KHALIL • INSERM U781, Hôpital Necker-Enfants Malades, Paris, France

GUILHERME MATTOS JARDIM COSTA • Laboratory of Cellular Biology,

NICHOLAS JOHN KUYPERS • Department of Anatomical Sciences and Neurobiology,

HALYNA R. SHCHERBATA • Max Planck Research Group of Gene Expression

Immunostaining of Germline Stem Cells and the Niche

Key words Drosophila, Germline stem cells, Niche, Immunostaining

For Drosophila germline stem cells (GSCs), the decision to undergo

enzyme such as alkaline phosphatase (AP) or horseradish peroxi-

2.1 10 M NaOH Weigh 4 g sodium hydroxide (NaOH) and dissolve them in 10 ml

2.3 PBT Add 1 ml Triton X-100 to 1,000 ml PBS (prepared in

2.4 20 % PFA Weigh 20 g paraformaldehyde powder, and then add 200 μl 10 M

2.7 3 % Bovine Weigh 30 g bovine serum albumin (BSA) and dissolve it in

2.9 Equipment Labquake (Model NO.415220, Barnstead international Inc.).

2.10 Microscope LEICA MZ12.5 stereomicroscope.

Fig. 2 The schematic diagram of dissection

1. 20 % PFA is stable at 4 °C for 6 weeks, after which its fixing

3. Other fixative buffers such as 4 % formaldehyde solution and

15. We recommend using cy3-conjugated antibody (Jackson

This work was supported by Temasek Life Sciences Laboratory

Genetic, Immunofluorescence Labeling, and In Situ

Key words Drosophila, Testis, Ovary, Germline stem cells, Niches

subset of neighboring stromal cells and extracellular substrates.

d Arm 1B1 e FasIII f Zfh-1

a DAPI b SSC cyst FC

c Arm 1b1 VASA DAPI d hh-GFP e FasIII

each germarium contains three types of stem cells: GSCs, escort

Through asymmetric division, GSC produces self-renewing GSC,

Prepare and store all reagents at room temperature (unless other-

2.1 Culturing 1. Control and transgenic Drosophila lines.

7. Foam or cotton plugs.

2.3 Generation 1. Stocks for generating FLP-mediated recombination germline

2.4 Immunostaining 1. Phosphate-buffered saline (PBS): 130 mM NaCl, 7 mM

25. Light, fluorescent, and confocal microscope.

2.5 Collection, 1. Drosophila adult flies.

2.6 In Situ 1. Dissecting solution (Drosophila Ringer’s solution): See

10. 96-well plate.

3.1 Generation 1. Clones of mutant GSCs were generated by Flp-mediated

4. Transfer the males and females at 25 °C to fresh food vial every

3.4 Immunofluore- 1. Fix the tissues in 4 % formaldehyde for 30 min.

3.5 Collection, 1. Collect embryos on grape juice plates overnight.

1 h, wash, and in the place of secondary antibody use Fast

1. The Fast Red working solution should be filtered to remove

M.K.S. is supported by the Knight’s Templar Eye Foundation and

through regulating DE-cadherin-mediated cell by JAK-STAT signaling. Science 294:

Visualization of Adult Stem Cells Within Their Niches Using

The Drosophila ovarian stem cell niche is very well characterized

terminal filaments. A variety of different markers makes it possible to

2.2 Ovary Dissection 1. Ice block for immobilization of the flies.

2.3 Fixation 1. Fixing solution: 4 % formaldehyde in phosphate-buffered

2.4 Antibodies 1. Blocking solution: 0.2 % bovine serum albumin, 5 % normal

Name of the Antibody Used to mark in the