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.
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).
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
|dc.relation.hasversion||Tura, O., E. M. Skinner, G. R. Barclay, K. Samuel, R. C. Gallagher, M. Brittan, P. W. Hadoke, D. E. Newby, M. L. Turner, and N. L. Mills. 2013. Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow. Stem Cells 31:338-348.||en