Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Abstract

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

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.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    CAS  Article  Google Scholar 

  2. Yamahara, K. et al. Augmentation of neovascularization in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. PLoS ONE 3, e1666 (2008).

    Article  Google Scholar 

  3. Sone, M. et al. Pathway for differentiation of human embryonic stem cells to vascular cell components and their potential for vascular regeneration. Arterioscler. Thromb. Vasc. Biol. 27, 2127–2134 (2007).

    CAS  Article  Google Scholar 

  4. Lu, S.J. et al. Generation of functional hemangioblasts from human embryonic stem cells. Nat. Methods 4, 501–509 (2007).

    CAS  Article  Google Scholar 

  5. Goldman, O. et al. A boost of BMP4 accelerates the commitment of human embryonic stem cells to the endothelial lineage. Stem Cells 27, 1750–1759 (2009).

    CAS  Article  Google Scholar 

  6. Nourse, M.B. et al. VEGF induces differentiation of functional endothelium from human embryonic stem cells: implications for tissue engineering. Arterioscler. Thromb. Vasc. Biol. 30, 80–89 (2009).

    Article  Google Scholar 

  7. Bai, H. et al. BMP4 regulates vascular progenitor development in human embryonic stem cells through a smad-dependent pathway. J. Cell Biochem. published online, doi:10.1002/jcb.22410 (30 November 2009).

  8. Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J. & Keller, G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432, 625–630 (2004).

    CAS  Article  Google Scholar 

  9. Levenberg, S., Zoldan, J., Basevitch, Y. & Langer, R. Endothelial potential of human embryonic stem cells. Blood 110, 806–814 (2007).

    CAS  Article  Google Scholar 

  10. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    CAS  Article  Google Scholar 

  11. Inman, G.J. et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol. Pharmacol. 62, 65–74 (2002).

    CAS  Article  Google Scholar 

  12. Gehling, U.M. et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood 95, 3106–3112 (2000).

    CAS  PubMed  Google Scholar 

  13. Kelly, M.A. & Hirschi, K.K. Signaling hierarchy regulating human endothelial cell development. Arterioscler. Thromb. Vasc. Biol. 29, 718–724 (2009).

    CAS  Article  Google Scholar 

  14. Peichev, M. et al. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 95, 952–958 (2000).

    CAS  PubMed  Google Scholar 

  15. Rafii, S. & Lyden, D. Cancer. A few to flip the angiogenic switch. Science 319, 163–164 (2008).

    CAS  Article  Google Scholar 

  16. Gao, D. et al. Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis. Science 319, 195–198 (2008).

    CAS  Article  Google Scholar 

  17. Lyden, D. et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat. Med. 7, 1194–1201 (2001).

    CAS  Article  Google Scholar 

  18. Rossig, L. et al. Histone deacetylase activity is essential for the expression of HoxA9 and for endothelial commitment of progenitor cells. J. Exp. Med. 201, 1825–1835 (2005).

    Article  Google Scholar 

  19. Ruzinova, M.B. & Benezra, R. Id proteins in development, cell cycle and cancer. Trends Cell Biol. 13, 410–418 (2003).

    CAS  Article  Google Scholar 

  20. Placantonakis, D.G. et al. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells 27, 521–532 (2009).

    CAS  Article  Google Scholar 

  21. Watabe, T. et al. TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells. J. Cell Biol. 163, 1303–1311 (2003).

    CAS  Article  Google Scholar 

  22. Jankovic, V. et al. Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 104, 1260–1265 (2007).

    CAS  Article  Google Scholar 

  23. Kang, Y., Chen, C.R. & Massague, J. A self-enabling TGFbeta response coupled to stress signaling: Smad engages stress response factor ATF3 for Id1 repression in epithelial cells. Mol. Cell 11, 915–926 (2003).

    CAS  Article  Google Scholar 

  24. James, D., Noggle, S.A., Swigut, T. & Brivanlou, A.H. Contribution of human embryonic stem cells to mouse blastocysts. Dev. Biol. 295, 90–102 (2006).

    CAS  Article  Google Scholar 

  25. Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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). https://doi.org/10.1038/nbt.1605

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1605

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing