ISOLATION AND CHARACTERIZATION OF UMBILICAL CORD BLOOD-DERIVED

MULTIPOTENT STEM CELLS ARISING FROM ADHERENT CD45+/CD34+ CELL SUBSET

David H McKenna, MD1,2,3, Sheryl D Adams, MT (ASCP)2, Eileen Emrick, MT (ASCP)2, Stacey Sprague, MT (ASCP)4, Barbara M Tigges, PhD4 and Daniel P Collins, PhD4
Medicine and Pathology, University of Minnesota, Minneapolis, MN; 2Clinical Cell Therapy Laboratory, University of Minnesota Medical Center; 3American Red Cross Cord
Blood Program; and 4BioE, Inc., Saint Paul, MN

1Laboratory

CONCLUSIONS

BACKGROUND
Conflicting data in the literature initially led to debate over the
presence of mesenchymal stem cells (MSCs) in umbilical cord
blood (UCB).1-3 UCB has since become an accepted source of
MSCs, stem cells capable of differentiating into cells of
connective tissue lineage.4 More recently UCB has been
examined for the presence of cells capable of differentiating
into cell types of all three embryonic layers (i.e., endo-, ecto-,

a)

b)

c)

d)

Figure 1. MLPCs early in culture with expression of: a) CD45 (day 6);
b) CD34 (day 6); c) CD133 (day 11); and d) SSEA-3 (day 11).

Figure 2. MLPCs in early fibroblastic stage of culture.

a)

b)

c)

d)

e)

f)

g)

h)

and meso-derm). Few groups have reported success in the

literature.5,6
Using a cell separation medium (PrepaCyte-MLPC, BioE,
Inc.) and plastic adherence, UCB-derived multipotent stem
cells were isolated, expanded, and characterized. These stem
cells, termed multi-lineage progenitor cells (MLPCs), were
able to give rise to cell types of the three embryonic layers.
MLPCs are the only such cell type from any source
demonstrated to be CD45+/CD34+ upon initial isolation.

METHODS
PrepaCyte-MLPC, an antibody-based cell separation medium,
is added to an UCB unit (American Red Cross Cord Blood
Program, Saint Paul, Minnesota USA). Following homo- and
heterophilic aggregation of undesired cell populations and
subsequent sedimentation to gravity, the supernatant
containing stem cells is expressed. After overnight incubation
in a T-flask in MSCGM (Cambrex, Inc.), non-adherent cells
are washed, leaving adherent cells to expand in culture. As
MLPC colonies are observed, cells are further enriched by
detachment (PBS/0.1% EGTA) and transfer to a new T-flask
(generally at 60-70% confluence). For differentiation assays,
cultures are typically grown to 80-90% confluence prior to
addition of special differentiation media.

UCB-derived MLPCs were isolated as a rare population of

adherent, initially CD45+/CD34+ cells with lymphoid
morphology. Following successful expansion in culture (5
of 16; 31%), multi-potency was demonstrated. Additional
differentiation studies (e.g., cardiac, hematopoiesis,
epithelial) are underway, and further pre-clinical testing
(e.g., animal teratoma studies) is planned. MLPCs may
serve a role in bone marrow transplantation (for malignant
and non-malignant disease) and regenerative medicine (e.g.,
cardiac muscle repair in acute/chronic cardiac disease, islet
cell replacement in diabetes mellitus); clinical studies are in
an early phase of development.

Figure 3. Differentiation of MLPCs: a) adipose (phase contrast); b) adipogenic (AdipoRed); c) myogenic (alpha-actinin); d)
myogenic (fast skeletal myosin); e) endothelial differentiation (E-selectin); f) endothelial differentiation (ICAM-2); g) neural
differentiation (beta tubulin III); h) hepatic differentiation (human serum albumin).

RESULTS
REFERENCES
MLPCs were isolated and expanded from 5 of 16 UCB units. Cells
initially possessed a lymphoid morphology and were positive for CD45,
CD34, CD133, SSEA-3, SSEA-4, and several MSC markers (see Figure
1). Several days into culture, expression of HSC/ESC markers was lost.
MSC marker expression was maintained; cells remained positive for
CD9, CD13, CD29, CD44, CD73, CD90, and CD105 and were
characterized by a fibroblastic morphology (see Figure 2). MLPC lines
have been cultured through greater than 20 passages with no apparent
impact on expansion or differentiation potential. Cell lines have been
successfully differentiated into cell types representative of each
embryonic layer (i.e., adipocytes, osteocytes, myocytes, endothelial cells,
hepatocytes, and neural cells). See Figure 3. Using limited-dilution
cloning techniques several clonal lines have been established. MLPCs
appear stable by karyotype, and they demonstrate expression of high
levels of genes associated with primitive, uncommitted, undifferentiated
stem cells (TERT, OCT-4, SOX-2, GATA-4, PTEN, PUM-2, TBX-3).

1. Erices A, Conget P, Minguell J. Mesenchymal progenitor

cells in human umbilical cord blood. Br J Haematol. 2000;
109: 235-242.
2. Goodwin H, Bicknese A, Chien S, et al. Multilineage
differentiation activity by cells isolated from umbilical cord
blood: expression of bone, fat, and neural markers. Biol
Blood Marrow Transplant. 2001; 7: 581-588.
3. Mareschi K, Biasin E, Piacibello W, et al. Isolation of
human mesenchymal stem cells: bone marrow versus
umbilical cord blood. Haematologica. 2001; 86: 1099-1100.
4. Bieback K, Kern S, Kluter H, and Eichler H. Critical
parameters for the isolation of mesenchymal stem cells
from umbilical cord blood. Stem Cells. 2004; 22: 625-634.
5. Lee OK, Kuo TK, Chen WM, et al. Isolation of
multipotent mesenchymal stem cells from umbilical cord
blood. Blood. 2004; 103: 1669-1675.
6. Kogler G, Sensken S, Airey J, et al. A new human somatic
stem cell from placental cord blood with intrinsic
pluripotent differentiation potential. J Exp Med. 2004; 200:
123-135.