Abstract 5439: Human Induced Pluripotent Stem Cell-derived Endothelial Cells Exhibit Heterogeneity
Vascular endothelial cells derived from pluripotent stem cells are useful in various therapeutic strategies for repair and revascularization of ischemic tissue in patients with vascular defects. One potential source for these therapeutic endothelial cells (ECs) is the induced pluripotent stem cells (iPSCs). In the present study, we generated iPSCs from human adult dermal fibroblasts using retrovirus-mediated transduction of Oct-4, Sox-2, Klf-4 and c-Myc. Differentiation to endothelial lineage was initiated by growing iPSCs in suspension for 4 days to form embryoid bodies (EB) and seeding on gelatin-coated dishes in αMEM and 20% defined FBS supplemented with 50ng/ml VEGF, bFGF and BMP4 for 10 days. iPSC-ECs were then isolated using phycoerythrin-conjugated CD31 antibody and were FACS sorted. Real time gene expression analysis and immunocytochemical staining of EC, pluripotency and transgene markers were performed. Further in vitro functional characterization such as matrigel tube formation, BrdU proliferation and cell migration assay in response to angiogenic growth factors were also carried out. All iPSC-ECs exhibited cobble-stone morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of iPSC-ECs revealed positive staining for EC markers such as CD31, vascular endothelial cadherin (CD144), endothelial nitric oxide synthase (eNOS) and von Willebrand factor (vWF). The iPSC-ECs proliferated well in growth factor-supplemented culture media and exhibited angiogenic behavior, forming capillary-like structures within 24 hours when placed in Matrigel. Intriguingly, we observed variations in the efficiency of endothelial generation between iPSC lines derived from different individuals, with EC yields ranging from 7±3.2% to 13±5.5% of total EB-derived cells. In addition, we noted that iPSC-ECs expressed lower levels of CD144, eNOS and vWF by rtPCR and immunohistochemistry when compared to human microvascular ECs. The results in this study demonstrated the feasibility of differentiating and isolating ECs from iPSCs. They also outline differences that exist between iPSC-EC lines and mature ECs. These differences may have an impact on functional capabilities of iPSC-ECs in cellular therapies.