Evaluation of functional roles of BMP2 and

Nanog in skeletal progenitor and in vivo

Manish Bais DVM, PhD

 Bone Biology

Bone consists of three cell types:

• Osteoblasts- formation of
new bone matrix
•Osteoclasts- antagonistic
function to osteoblasts,
resorb already present
bone matrix
•Osteocytes- osteoblasts
that have become
enclosed within bone
matrix and no longer
deposit matrix
MSC differentiation to osteogenic lineage

Osteocalcin,
Alkaline
phosphatase,
Runx2,
Dlx5, Osterix

Ex- vivo
Ascorbate, β-glycerol phosphate, dex
MSCs

Day 0 4 6……………………………………………………………..21
Transcription factors network in osteoblastogenesis

Dlx3
• BMP2 function in skeletal lineage

• Induction of ES cell related gene, Nanog in bone injury and

its functional role

• Rath Peptide based delivery system

Introduction:

Combined gene transfer of BMP2 and BMP7 induces

stable spinal fusion in rodent model (Zhu et al., 2004)
 Hypothesis:

BMP2 function is indispensable in MSCs and bone injury

which could not be rescued by other osteoinductive protein.

BMP2 does not affect stem cell recruitment.

 VSV-G pseudotyped lentivirus can tranduce MSCs
and loss of BMP2 affect loss of ostegenesis

PLK0-GFP shBMP2

C= control
NT= NT shRNA

Toxicity

Alizarin Red
staining
Can the phenotype be rescued with other osteoinductive
protein- BMP7 ?
 Regulation of transcription factor network by BMP7

Time course induction Phenotype

in osteogenic lineage rescue
Exogenous BMP2 rescue phenotype downregulated by
shBMP2
Bone marrow ablation model to study gene function

• Lentivirus mediated efficient

method to study the particular
gene function in vivo as a model

• Destroy the niche and allow it to

regenerate once again
Efficient transduction of lentivirus expressing gene in vivo

EF1α-Luc

CMV-LacZ
Lentiviral shBMP2 diminishes trabecular bone formation

Micro-CT
Loss of BMP2 blocks regeneration of new bone formation
BMP2 affects downstream transcription factors
 BMP2 does not affect stem cell recruitment

Bais MV, Wigner N, Young M, Toholka R, Graves DT, Morgan EF, Gerstenfeld
LC and Einhorn TA BMP-2 Is Essential for Post Natal Osteogenesis but not
for Recruitment of Osteogenic Stem Cells (Manuscript : Bone).
 Interim conclusion

Loss of endogenous BMP2 could not completely

rescued by BMP7

BMP2 is not essential for stem cell recruitment and acts

downstrem of stem cell population
Inducible expression of BMP2 in transgenic mouse
model
Inducible expression of BMP2 in transgenic mouse
model to study role in different stages of healing

Ariad Inc.

1. Activation of gene expression is controlled by the induced binding of

transcription factor proteins to target genes.
2. Transcription factors are bifunctional proteins that recognize specific DNA
sequences near target genes and cause transcription initiation.
3. Sequence specific transcriptional activator expressed as individual proteins
and brought together via a noncovalent interaction and induced by rapamycin
analog AP21967.
 Components of inducible system

Transcription Factor Inducible Target Gene

Constructs Constructs
1. Constitutive (CMV) 2. Indicator (luciferase)
2. Tissue specific 3. BMP-2
(aggrecan/OC)
Inducible luciferase expression Ex-vivo and in vivo
Day 7
The skin fibroblast from C Rapamycin C Rapamycin
transgenic mice transfected
with CMV-TF construct or
inducible luciferase construct,
transiently

30000
CMV-TF mice
20000

10000

0 Day 21
No diamerizer 5uM dimerizer
AP21967 AP21967
3000 CMV-TF X Ind – Luc mice

2000
1000000 Ind-Luc mice

500000 1000

0 0
No diamerizer 5uM dimerizer control bone spleen kidney lung liver mix all
AP21967 AP21967 tissues
Inducible BMP2 expression produces specific
phenotype- like osteoporosis??
Non-injured bone

0.1
Connection density
Fracture callus

0.05 3 Human BMP2 osteocalcin

Runx2 osterix
Dlx5 Msx2
Rapalolg (AP21967) n = 4
0

vehicle treated rapalog treated 2

Trabecular thickness
100 1

50

0 0
vehicle tr eated r apamycin tr eated BMP2 X CMV control- No Transgenic BMP2 with
Rapamycin (n=5 ) rapamycin (n=5 )
Conclusions:

Rapamycin (AP21967) based inducible system is active

in in vivo and could be used for trangenic mice
generation.

Inducible BMP2 expression induces phenotype similar

to osteoporotic bone in noninjured bone whereas
upregulate osterix, Runx2, Dlx5 and downregulate
osteocalcin and noggin in bone injury.
 Traumatic injury model
Closed femoral fractures

Blunt Trauma Induced

Fixed by intrameduallary
Roding
Tissues and cell type that contribute to repair

Initial Injury Endochondral Phase Primary Bone Formation Secondary Bone Formation

1-3 days 3-10 days 3-14 days 14-49 days

Inflammation Periosteal Response Bone Cell Recruitment Establishment of Marrow
Hematoma Formation Vascular In-growth Chondrocyte Apoptosis Osteoclast Remodeling
Marrow Response Intramembraneous Matrix Proteolysis Coupled Osteoblast Recruitment
MSC Recruitment Bone Osteoclast* Recruitment
Formation Endochondral Neo-Vascularization
 Transcriptome analysis of fracture healing
Up Down Variable
Proteolysis
4.4%
Cell Adhesion Lymphocyte 1.4%
4.1% Gametogenesis RNA processing 0.2% Neurogenesis Signaling
Skeletal 1.9% Catabolism 3.5% 2.1%
4.5% Miscellaneous 10.5% 12.5% Cell Cycle 3.1%
1.4% 1.9% Miscellaneous
7.7% Signaling 4.9% 0.3% T-Cells 1.2% 10.7%
Vasculogenesis
5.2% 4.4% Stress Response 11.6%
20% Development 6.7%
Cell Cycle Development
5.8% 14.5%
14.9%Hematopoiesis 1.8%
Transcription
12.7% 6.5% Motility Metabolism
2.2% 3% Immune 11.7% 7.3%
Cyto/Actin
2.3% 8.5% Ion Transport 3.8% Inflammation
Neurogenesis 1.9%
Immune 8% Metabolism 17.7% 1.9% Apoptosis 4.3% Motility Antigen
4.9%
Apoptosis 2% DNA Repair 19.4% 3.1%
Replication
3.1% 1.1% 5.1%
Ion Transport Transcription Cyto/Actin
5.3% 1% 1.8%

N=7141 N=2876 N=1292

N= total number of expressed mRNAs in a group

Bais MV, McLean J, Sabastiani P,Young M, Wigner N, Smith T, Kotton DN,

Warman M, Einhorn TA, Gerstenfeld LC Transcriptional Analysis Of
Fracture Healing And The Induction Of Embryonic Stem Cell-Related
Genes During Fracture Healing. ( Revised: Plos One)
Transcriptional Profile of Stems Cells

Meta analysis of literature of ESC, MSC, NSC, HSC

Identification of ~1076 Common Genes Associated with
“Stemness” of these Lineages

425 of these Genes Observed in the Clusters

139 of these Genes Observed in 6 clusters of these

clusters 5 overlap 3 of four Lineages (Endothelia,
Skeletolgenic, Neurogenic and Myogenic Lineages)
 Nanog Is Expressed During Both Fracture
Healing and MSC Osteogneic Differentiation.

mFx: Nanog mMSC: Nanog

60 4

Fold Induction Over d10

Fold Induction Over d0

30 2

0 0
0 1 3 5 7 10 14 10 12 14 16 21
Days After Fracture Days After Plating
 Nanog gene is functional in post-natal cells
Fold change nanog mRNA

1.5
5' nanog promoter (2.5Kb) active in W20-17 cells

RLU- relative luminence unit of lucifease

1.06
1 10 sec 1 min 10 min
30000

0.5
20000

activity
0.07
0
NTshRNA Nanog 336 shRNA 10000

0
Fold change osteocalcin mRNA

l1

l2

l3

3
st

st

st
ro

ro

ro

te

te

te
nt

nt

nt
co

co
co
8

0
NT shRNA Nanog 336 shRNA
Loss of Nanog upregulate the genes related to
differentiation in vivo at day 7

Nanog expression smad1

sox9 osterix
Fold change mRNA
Dlx5 Runx2
osteocalcin
4.00

3.00

2.00

1.00

0.00
Nanog shRNA NT shRNA

Nanog shRNA NT shRNA

Loss on Nanog affects bone formation in
vivo- Micro-CT analysis
Identification of Nanog positive cells in
vivo in traumatic injury
Characterization of Nanog expressing
cells from transgenic mice

Riken,
Japan

Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced

pluripotent stem cells. Nature. 2007 19;448(7151):313-7.
Conclusion:

Nanog has functional role in post-natal cells and

tissue
Loss of nanog leads loss of bone formation in bone
marrow ablation model

Question:

Nanog positive cells belong to which lineage:

Hematopoiteic or mesenchymal?
Grafting potential for regeration of tissuses?
 Nonviral delivery system

Identified Cell penetrating peptide from VP5 protein

Infectious Bursal disease virus of poultry.

Peptide based delivery system using 24 amino acid

peptide

Has hydrophilic and hydrophobic domain

 Rath peptide based delivery of oligonucleotide, antibody
and plasmid
Rath peptide-FITC

Oligonuclotide-
vero cells

Antibody- vero cells

Antibody- CEFs
primary cells

CMV-GFP plasmid
vero cells

Bais MV, Kumar S, Tiwari AK, Kataria RS, Nagaleekar VK, Shrivastava S, Chindera K. Novel Rath peptide for intracellular
delivery of protein and nucleic acids.Biochem Biophys Res Commun. 2008 May 23;370(1):27-32. Epub 2008 Mar 17.
PMID: 18346454.
 Structural characterization of Rath peptide

CD spectroscopy

Fluorescence
spectroscopy

Transmission
Electron
Microscopy
Orthopedic Surgery Lab

Thomas Einhorn, MD
Louis Gerstenfeld, PhD
Jody McLean, BS
Zabrina Shabin, BS
Nathan Wigner , BS (MD/PhD)
Megan Yang, MD

Collaborators:
Philip Trackman, PhD
Darrell Kotton, MD
Temple Smith, PhD
Dana Graves, DMD, DMSc
Elise Morgan, PhD