Pluripotent stem cell-derived endothelial cells for vascular regeneration

Abstract

Background: Vascular endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. As such, the study of endothelial cells has sought to identify causal pathways and novel therapeutic approaches to promote vascular repair. Induced pluripotent stem (iPS) cell technology may be a particularly useful tool, and could be used to derive endothelial cells and their progenitors from individuals with endothelial dysfunction to explore these pathways and develop novel strategies for vascular regeneration. Whilst iPS cells are conventionally obtained from the reprogramming of dermal fibroblasts, it was hypothesised that endothelial cells could also be reprogrammed, and that these pluripotent cells would have enhanced capacity for endothelial differentiation and vascular regeneration. Objectives: To generate iPS cells from human fibroblasts and endothelial cells and to assess their potential for endothelial differentiation and vascular regeneration. Methods and Results: A) Reprogramming: Dermal fibroblasts and endothelial outgrowth cells from blood were obtained from healthy donors (n=5) and transfected with episomal vectors containing six reprogramming factors: Sox2, Klf4, Oct3/4, L-Myc, Lin28 and Shp53. Successfully reprogrammed fibroblast-derived iPS (fiPS) and endothelial cell-derived iPS (eiPS) arose as colonies, and were isolated and expanded. Reprogrammed cells expressed pluripotency markers SSEA3, SSEA4, TRA 1 60, Oct3/4 and NANOG, and developed into all three germ layers following embryoid body formation. B) Endothelial differentiation: iPS and ES cell lines were aggregated into embryoid bodies in stem cell growth media containing mesoderminducing cytokines. Embryoid bodies were then disaggregated and cultured in endothelial medium supplemented with VEGF. After seven days, a population of CD31+ cells was isolated and further cultured. Mature endothelial cell antigen expression was confirmed by flow cytometry. CD31+ cells were similar to mature endothelial cells in functional assays of proliferation, migration, nitric oxide production and angiogenesis. C) Comparison of fiPS versus eiPS: eiPS differentiated into endothelial cells with greater efficiency than fiPS (21±3% versus 3±2%, P<0.05). fiPS-derived endothelial cells and eiPS-derived endothelial cells expressed similar levels of endothelial markers CD146, CD31, VEFGR2 and CD34 compared to control endothelial cells. When grown on Matrigel, they formed tubule-like structures with a similar number of vessel connections. In vivo, endothelial cells derived from fiPS and eiPS increased neovasculogenesis in a nude mouse model: vessel density was increased after implantation of endothelial cells from fiPS and eiPS by 3.50 vessel counts (P≤0.001) and 3.47 vessel counts (P≤0.001) respectively, when compared to controls. By comparison control endothelial cells did not increase vessel density compared to control (P>0.05). Conclusions: Endothelial cells can be isolated from blood and reprogrammed to form pluripotent stem cells with enhanced capacity to differentiate into endothelial cells than those derived from dermal fibroblasts. Endothelial cells derived from both sources promote angiogenesis in vivo, and have major potential for therapeutic applications in vascular regeneration.