1. Aim: Mesenchymal stem cells (MSCs) have the ability to differentiate into mul-
tiple cell lineages. The differentiation of bone marrow derived MCSs to endo-
thelial cells (ECs) have been reported. However, the differentiation of adipose
derived MSCs from different types of adipose tissue into ECs has not been in-
vestigated. In this study, we investigated the endothelial differentiation of
MSCs isolated from Subcutaneous and Omentum/Visceral adipose tissue.
Methods: MSCs isolated from Porcine adipose tissue were characterized by
positive staining for MSC markers CD44, CD90, CD105 and negative staining
for macrophage marker CD11b and hematopoietic stem cell marker CD45.
The MSCs were stimulated with endothelial growth media (EGM-2 Lonza con-
taining 50ng of VEGF) for 10 days. The differentiated MSCs were assessed
for mRNA expression by qPCR (n=3) and protein expression by flow cytometry
(n=3) for endothelial cell markers: Von Willebrand factor (vWF), vascular endo-
thelial-cadherin (VE-cadherin) and platelet endothelial cell adhesion molecule
(PECAM1) following differentiation. Angiogenesis assay (n=3) and acetylated-
low density lipoprotein uptake assay (LDL uptake) (n=3) were used to assess
the endothelial functionality.
Results: Porcine adipose derived MSCs from Subcutaneous adipose tissue
show an increase of mRNA expression for vWF(p≤0.01), VE-cadherin(p≤0.01),
PECAM1(p≤0.05), after 10 day differentiation. MSCs from Subcutaneous adi-
pose tissue demonstrated higher number of vWF, VE-cadherin and PECAM1
positive cells after 10 day differentiation. Angiogenesis assay showed a signifi-
cant increase in capillary tube sprouting and polygon formation with the differ-
entiated MSCs derived from Subcutaneous adipose tissue. Fluorescent evalu-
ation of the LDL uptake assay showed increase of staining with the differentiat-
ed MSCs derived form Subcutaneous tissue.
Conclusion: Porcine MSCs derived from Subcutaneous adipose tissue
demonstrated enhanced endothelial differentiation when compared to differen-
tiated MSCs derived form Omentum adipose tissue. This shows that MSCs
derived form Subcutaneous adipose tissues are more suitable option for re-
generating the endothelial layer limiting intimal hyperplasia.
This work was supported by the National Institutes of Health/National Heart, Lung, and Blood Institute
Grant 1R01HL120659 to D.K.A.
ABSTRACT
SUMMARY OF THE RESULTS
Porcine MSCs derived form Subcutaneous adipose tissue demonstrated enhanced endothelial differenti-
ation when compared to differentiated MSCs derived from Omentum adipose tissue. This shows that
MSCs derived form subcutaneous adipose tissue are more suitable option for regenerating the endotheli-
al layer limiting intimal hyperplasia
Endothelial differentiation of Subcutaneous and Omentum Porcine Adipose Derived Mesenchymal Stem Cells: A Comparison
Jaideep Sahni1
, Yovani Llamas1,2
, Divya Pankajakshan1
, and Devendra K. Agrawal1,2
Center for Clinical and Translational Science1
, Department of Medical Microbiology and Immunology2
,
Creighton University School of Medicine, Omaha, NE, USA
ACKNOWLEDGEMENTS
CONCLUSION
Figure 4: Angiogenesis assay. MSCs in culture (1); HUVEC
(A), Subcutaneous derived MSCs after 10 day differentiation
(B), Omentum derived MSCs after 10 day differentiation (C),
Subcutaneous derived MSCs (D), Omentum derived MSCs
(E). At 40x magnification.
Characterization of mesenchymal stem cells (MSCs) derived from porcine adipose tissue
Functional Assays: Endothelial differentiation of Subcutaneous and Omentum Porcine Adipose Derived Mesenchymal Stem Cells: Functional assays
Endothelial differentiation of Subcutaneous and Omentum Porcine Adipose Derived Mesenchymal Stem Cells – mRNA and protein expression of
endothelial cell markers
Figure 6: Protein Expression. Differentiated MSCs from both Omentum and Subcutaneous show a statis-
tically significant increase(p≤0.001) for VE-cadherin and PECAM.
Figure 3. LDL Uptake. Top row shows DAPI staining, middle row shows LDL fluorescence staining, bottom row shows combined DAPI and LDL fluores-
cence staining. At 40x magnification.
INTRODUCTION
Most noncommunicable disease (NCD) deaths are caused by cardiovascular
disease (17.3 million people annually); outranking deaths caused by cancers,
respiratory diseases, and diabetes. Endothelial denudation is a common occur-
rence during angioplasty procedures. Stem cell therapy with Mesenchymal
stem cells (MSCs) have been extensively used for tissue regeneration. This
makes the use of MSCs to facilitate intimal hyperplasia for post-angioplasty
procedures a captivating idea. The two extensively used sources of MSCs are
bone marrow and adipose tissue. The advantage of using adipose tissue as a
MSC source is that the isolation procedures are less invasive and less expen-
sive compared to bone marrow aspiration. Moreover the percentage of MSCs in
adipose tissue is higher than that of bone marrow. MSC phenotype might also
differ depending on specific areas of adipose tissue isolation.
In this study we investigated the endothelial differentiation of MSCs isolated
from Subcutaneous adipose tissue and Omentum/Visceral adipose tissue. The
MSCs were differentiated to endothelial cells (ECs) by exposing MSCs to endo-
thelial cell differentiating media (EGM-2 Lonza containing 50ng of VEGF) for 10
days. The cells were assessed for EC markers by mRNA and protein expres-
sion studies. Functional analyses of differentiating cells were done by angio-
genesis assay and acetylated low density lipoprotein uptake assay (LDL up-
take).
1. Flow cytometry data demonstrated positive expression of CD44, CD90, and CD105 and negative ex-
pression of CD11b, CD34, and CD45, which are characteristics of MSCs.
2. The overall mRNA expression levels of vWF, VE-cadherin and PECAM1 expression was significantly
greater for Subcutaneous MSCs after 10 day stimulation.
3. The protein expression levels of VE-cadherin and PECAM1 expression significantly increased for both
Omentum and Subcutaneous MSCs after 10 day stimulation.
4. Stimulated Subcutaneous MSCs showed a significant increase of capillary tube sprouting and polygon
formation in the Angiogenesis assay.
5. Stimulated Subcutaneous MSCs showed a significant increase in LDL uptake.
Differentiated
Omentum MSCs
Differentiated
Subcutaneous MSCs
Subcutaneous
MSCs
Omentum MSCs HUVEC
Figure 1. Characterization of Omentum MSCs. Immunophenotyping data
showing positive staining for CD44, CD105, CD90; negative staining of
CD11b, and CD45.
Figure 5: mRNA Expression. Differentiated MSCs from Omentum show a statistically
significant increase(p≤0.01) for VE-cadherin and PECAM. Differentiated Subcutaneous
MSCs show a statistically significant increase(p≤0.01) for vWF(p≤0.01) VE-
cadherin(p≤0.01) and PECAM1(p≤0.05).
Figure 2. Characterization of Subcutaneous MSCs. Immunophenotyping
data showing positive staining for CD44, CD105, CD90; negative staining of
CD11b, CD34 and CD45.