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Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent


Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

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Figure 1: Sequential TGFβ activation followed by inhibition during phase 1 differentiation promotes a tenfold expansion of hVPr-GFP+ hESC-derived cells.
Figure 2: TGFβ inhibition after endothelial cell isolation during phase 2 increases yield and preserves vascular identity of purified endothelial cells.
Figure 3: Molecular profiling of hESC-derived endothelial cells reveals a signature defined by high Id1 expression.
Figure 4: TGFβ inhibition upregulates Id1 expression and is necessary for the increased yield of functional endothelial cells capable of in vivo neo-angiogenesis.

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We thank A. Brivanlou for providing the RUES1 hESC line. D.J., M.S. and G.L. are Fiona and Stanley Druckenmiller Fellows of the New York Stem Cell Foundation. S.R. is supported by Howard Hughes Medical Institute; Ansary Stem Cell Institute; Anbinder and Newmans Own Foundation; National Heart, Lung, and Blood Institute R01 grants HL075234 and HL097797; Qatar National Priorities Research Program; and Empire State Stem Cell Board and New York State Department of Health, NYS C024180.

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Authors and Affiliations



D.J. designed and performed the experiments and wrote the manuscript. H.-s.N. and R.B. designed and created the Id1-YFP BAC transgenic vector. M.S. performed experiments and contributed to the manuscript. D.N. performed flow cytometric experiments. T.J. performed molecular cloning. M.T. and L.S. generated the Id1-YFP BAC transgenic hESC line. L.S. and G.L. generated the FD iPSC line. N.Z. and Z.R. generated the hESC lines WMC2, WMC8 and WMC9. D.L. and S.Y.R. designed experiments and performed data analysis. S.R. designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Shahin Rafii.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figs.1–5 and Supplementary Tables 1 (PDF 9976 kb)

Supplementary Video 1

Detection of vasculogenesis in Real-Time: hVPr-GFP+ cells appear at day 5 and connect to form primitive vascular tubules. (MOV 21888 kb)

Supplementary Video 2

Real-Time Tracking of hESC-derived ECs: Remodeling of hVPr-GFP+ vessel-like structures in adhering EBs (MOV 7553 kb)

Supplementary Video 3

Establishment of vascular patterning: Human VPr-GFP+ cells form branching microvascular structures with closed lumens. (MOV 7488 kb)

Supplementary Video 4

Tubulogenesis of human neo-vessels in vitro: Human VPr-GFP+ cells reorganize into large vessel-like structures following extendeddifferentiation in vitro. (MOV 3244 kb)

Supplementary Video 5

Whole-well immunodetection of non-endothelial cell types that emerge from hESC-derived ECs in the absence of TGF inhibition. (MOV 9813 kb)

Supplementary Video 6

Whole-well immunodetection of mitotic hESC-derived ECs in the absence of TGF inhibition. (MOV 58218 kb)

Supplementary Video 7

Whole-well immunodetection of mitotic hESC-derived ECs in the presence of TGF inhibition. (MOV 67678 kb)

Supplementary Video 8

Human ESC-derived ECs cultured in the presence of TGF inhibitor form functional vessels in vivo. (MOV 8313 kb)

Supplementary Video 9

Human ESC-derived ECs cultured in the presence of TGF inhibitor form functional vessels in vivo. (MOV 14639 kb)

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James, D., Nam, Hs., Seandel, M. et al. Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent. Nat Biotechnol 28, 161–166 (2010).

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