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.