Skip to main content

Thank you for visiting 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.

Direct conversion of human amniotic cells into endothelial cells without transitioning through a pluripotent state


Endothelial cells (ECs) have essential roles in organ development and regeneration, and therefore they could be used for regenerative therapies. However, generation of abundant functional endothelium from pluripotent stem cells has been difficult because ECs generated by many existing strategies have limited proliferative potential and display vascular instability. The latter difficulty is of particular importance because cells that lose their identity over time could be unsuitable for therapeutic use. Here, we describe a 3-week platform for directly converting human mid-gestation lineage-committed amniotic fluid–derived cells (ACs) into a stable and expandable population of vascular ECs (rAC-VECs) without using pluripotency factors. By transient expression of the ETS transcription factor ETV2 for 2 weeks and constitutive expression the ETS transcription factors FLI1 and ERG1, concomitant with TGF-β inhibition for 3 weeks, epithelial and mesenchymal ACs are converted, with high efficiency, into functional rAC-VECs. These rAC-VECs maintain their vascular repertoire and morphology over numerous passages in vitro, and they form functional vessels when implanted in vivo. rAC-VECs can be detected in recipient mice months after implantation. Thus, rAC-VECs can be used to establish a cellular platform to uncover the molecular determinants of vascular development and heterogeneity and potentially represent ideal ECs for the treatment of regenerative disorders.

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

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: The rAC-VEC platform is a 3- to 4-week reprogramming process that requires the transduction of specific transcription factors (TFs).
Figure 2: Morphology of freshly isolated and precultured amniotic cells.
Figure 3: ACs transduced with ETS-TFs express multiple EC markers within 3–4 weeks.


  1. Ding, B.S. et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310–315 (2010).

    Article  CAS  Google Scholar 

  2. Ding, B.S. et al. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell 147, 539–553 (2011).

    Article  CAS  Google Scholar 

  3. Hu, J. et al. Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416–419 (2014).

    Article  CAS  Google Scholar 

  4. Butler, J.M. et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251–264 (2010).

    Article  CAS  Google Scholar 

  5. Ding, L., Saunders, T.L., Enikolopov, G. & Morrison, S.J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).

    Article  CAS  Google Scholar 

  6. Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat. Cell Biol. 12, 1046–1056 (2010).

    Article  CAS  Google Scholar 

  7. Rafii, S. et al. Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 86, 3353–3363 (1995).

    CAS  PubMed  Google Scholar 

  8. Rafii, S. et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 84, 10–19 (1994).

    CAS  Google Scholar 

  9. Nolan, D.J. et al. Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration. Dev. Cell 26, 204–219 (2013).

    Article  CAS  Google Scholar 

  10. Sandler, V.M. et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 312–318 (2014).

    Article  CAS  Google Scholar 

  11. Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).

    Article  CAS  Google Scholar 

  12. Choi, K.D. et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27, 559–567 (2009).

    Article  CAS  Google Scholar 

  13. James, D. et al. Expansion and maintenance of human embryonic stem cell-derived endothelial cells by TGF-β inhibition is Id1 dependent. Nat. Biotechnol. 28, 161–166 (2010).

    Article  CAS  Google Scholar 

  14. Prasain, N. et al. Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells. Nat. Biotechnol. 32, 1151–1157 (2014).

    Article  CAS  Google Scholar 

  15. Morita, R. et al. ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells. Proc. Natl. Acad. Sci. USA 112, 160–165 (2015).

    Article  CAS  Google Scholar 

  16. Kurian, L. et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat. Methods 10, 77–83 (2013).

    Article  CAS  Google Scholar 

  17. Lagarkova, M.A., Volchkov, P.Y., Philonenko, E.S. & Kiselev, S.L. Efficient differentiation of hESCs into endothelial cells in vitro is secured by epigenetic changes. Cell Cycle 7, 2929–2935 (2008).

    Article  CAS  Google Scholar 

  18. Wang, Z.Z. et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels. Nat. Biotechnol. 25, 317–318 (2007).

    Article  CAS  Google Scholar 

  19. Kane, N.M. et al. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and. Arterioscler. Thromb. Vasc. Biol. 30, 1389–1397 (2010).

    Article  CAS  Google Scholar 

  20. Levenberg, S., Golub, J.S., Amit, M., Itskovitz-Eldor, J. & Langer, R. Endothelial cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 4391–4396 (2002).

    Article  CAS  Google Scholar 

  21. Ginsberg, M. et al. Efficient direct reprogramming of mature amniotic cells into endothelial cells by ETS factors and TGF-β suppression. Cell 151, 559–575 (2012).

    Article  CAS  Google Scholar 

  22. Pera, M.F. Stem cells: the dark side of induced pluripotency. Nature 471, 46–47 (2011).

    Article  CAS  Google Scholar 

  23. Mummery, C. Induced pluripotent stem cells—a cautionary note. N. Engl. J. Med. 364, 2160–2162 (2011).

    Article  CAS  Google Scholar 

  24. Okano, H. et al. Steps toward safe cell therapy using induced pluripotent stem cells. Circ. Res. 112, 523–533 (2013).

    Article  CAS  Google Scholar 

  25. Morris, S.A. et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell 158, 889–902 (2014).

    Article  CAS  Google Scholar 

  26. de Rham, C. & Villard, J. Potential and limitation of HLA-based banking of human pluripotent stem cells for cell therapy. J. Immunol. Res. 2014, 518135 (2014).

    Article  Google Scholar 

  27. Benavides, O.M., Petsche, J.J., Moise, K.J. Jr., Johnson, A. & Jacot, J.G. Evaluation of endothelial cells differentiated from amniotic fluid-derived stem cells. Tissue Eng. Part A 18, 1123–1131 (2012).

    Article  CAS  Google Scholar 

  28. De Coppi, P. et al. Isolation of amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100–106 (2007).

    Article  CAS  Google Scholar 

  29. Konig, J. et al. Amnion-derived mesenchymal stromal cells show angiogenic properties but resist differentiation into mature endothelial cells. Stem Cells Dev. 21, 1309–1320 (2012).

    Article  Google Scholar 

  30. Zhang, P., Baxter, J., Vinod, K., Tulenko, T.N. & Di Muzio, P.J. Endothelial differentiation of amniotic fluid-derived stem cells: synergism of biochemical and shear force stimuli. Stem Cells Dev. 18, 1299–1308 (2009).

    Article  CAS  Google Scholar 

  31. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  32. Markoulaki, S. et al. Transgenic mice with defined combinations of drug-inducible reprogramming factors. Nat. Biotechnol. 27, 169–171 (2009).

    Article  CAS  Google Scholar 

  33. Efe, J.A. et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222 (2011).

    Article  CAS  Google Scholar 

  34. Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl. Acad. Sci. USA 108, 7838–7843 (2011).

    Article  CAS  Google Scholar 

  35. Lindenmair, A. et al. Mesenchymal stem or stromal cells from amnion and umbilical cord tissue and their potential for clinical applications. Cells 1, 1061–1088 (2012).

    Article  CAS  Google Scholar 

  36. Lee, D. et al. ER71 acts downstream of BMP, Notch, and Wnt signaling in blood and vessel progenitor specification. Cell Stem Cell 2, 497–507 (2008).

    Article  CAS  Google Scholar 

  37. Liu, F., Walmsley, M., Rodaway, A. & Patient, R. Fli1 acts at the top of the transcriptional network driving blood and endothelial development. Curr. Biol. 18, 1234–1240 (2008).

    Article  CAS  Google Scholar 

  38. McLaughlin, F. et al. Combined genomic and antisense analysis reveals that the transcription factor Erg is implicated in endothelial cell differentiation. Blood 98, 3332–3339 (2001).

    Article  CAS  Google Scholar 

  39. De Val, S. & Black, B.L. Transcriptional control of endothelial cell development. Dev. Cell 16, 180–195 (2009).

    Article  CAS  Google Scholar 

  40. Birdsey, G.M. et al. Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. Blood 111, 3498–3506 (2008).

    Article  CAS  Google Scholar 

  41. De Val, S. et al. Combinatorial regulation of endothelial gene expression by ETS and forkhead transcription factors. Cell 135, 1053–1064 (2008).

    Article  CAS  Google Scholar 

  42. Dryden, N.H. et al. The transcription factor Erg controls endothelial cell quiescence by repressing activity of nuclear factor (NF)-κB p65. J. Biol. Chem. 287, 12331–12342 (2012).

    Article  CAS  Google Scholar 

  43. Kataoka, H. et al. Etv2/ER71 induces vascular mesoderm from Flk1+PDGFRα+ primitive mesoderm. Blood 118, 6975–6986 (2011).

    Article  CAS  Google Scholar 

Download references


We are indebted to D. James and V. R. Pulijaal (Weill Cornell Medicine) for help in providing material and intellectual input. W.S., K.S. and S.R. are supported by Ansary Stem Cell Institute, the Empire State Stem Cell Board and New York State Department of Health grants (C026878, C028117, C029156). S.R. is supported by the National Heart, Lung, and Blood Institute (R01HL115128, R01HL119872 and R01HL128158), the National Cancer Institute (U54CA163167), the National Institute of Diabetes and Digestive and Kidney Diseases (R01DK095039), the Qatar National Priorities Research Program (NPRP 6-131-3-268), and the Howard Hughes Medical Institute. W.S. was also supported by a US National Institutes of Health (NIH) training grant (T32HL94284).

Author information

Authors and Affiliations



S.R. envisioned the original idea. M.G., W.S. and S.R. developed the protocol and wrote the manuscript. M.G. performed the majority of the experiments. W.S. contributed to these experiments. W.S. and K.S. conceived the project and interpreted the data. K.S. wrote the IRB protocol and provided assistance in obtaining the AC samples. All authors commented on the manuscript.

Corresponding author

Correspondence to Shahin Rafii.

Ethics declarations

Competing interests

M.G. is a senior scientist with Angiocrine Bioscience. S.R. is a co-founder of Angiocrine Bioscience.

Integrated supplementary information

Supplementary Figure 1 rAC-VECs can be generated by a defined serum-free media supplemented with VEGF-A and FGF-2 cytokines.

Surface expression of VE-cadherin was assayed for by FACS at 4 days (i., iv.), 12 days (ii., v.), and 21 days (iii., vi.) post-lentiviral transduction with ETS-TFs. A minimum of 1 X 105 cells were analyzed for each sample.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Manual and Supplementary Table 1 (PDF 1064 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ginsberg, M., Schachterle, W., Shido, K. et al. Direct conversion of human amniotic cells into endothelial cells without transitioning through a pluripotent state. Nat Protoc 10, 1975–1985 (2015).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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