Abstract
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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ding, B.S. et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310–315 (2010).
Ding, B.S. et al. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell 147, 539–553 (2011).
Hu, J. et al. Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416–419 (2014).
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).
Ding, L., Saunders, T.L., Enikolopov, G. & Morrison, S.J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).
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).
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).
Rafii, S. et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 84, 10–19 (1994).
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).
Sandler, V.M. et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 312–318 (2014).
Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).
Choi, K.D. et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27, 559–567 (2009).
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).
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).
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).
Kurian, L. et al. Conversion of human fibroblasts to angioblast-like progenitor cells. Nat. Methods 10, 77–83 (2013).
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).
Wang, Z.Z. et al. Endothelial cells derived from human embryonic stem cells form durable blood vessels. Nat. Biotechnol. 25, 317–318 (2007).
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).
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).
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).
Pera, M.F. Stem cells: the dark side of induced pluripotency. Nature 471, 46–47 (2011).
Mummery, C. Induced pluripotent stem cells—a cautionary note. N. Engl. J. Med. 364, 2160–2162 (2011).
Okano, H. et al. Steps toward safe cell therapy using induced pluripotent stem cells. Circ. Res. 112, 523–533 (2013).
Morris, S.A. et al. Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell 158, 889–902 (2014).
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).
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).
De Coppi, P. et al. Isolation of amniotic stem cell lines with potential for therapy. Nat. Biotechnol. 25, 100–106 (2007).
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).
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).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
Markoulaki, S. et al. Transgenic mice with defined combinations of drug-inducible reprogramming factors. Nat. Biotechnol. 27, 169–171 (2009).
Efe, J.A. et al. Conversion of mouse fibroblasts into cardiomyocytes using a direct reprogramming strategy. Nat. Cell Biol. 13, 215–222 (2011).
Kim, J. et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc. Natl. Acad. Sci. USA 108, 7838–7843 (2011).
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).
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).
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).
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).
De Val, S. & Black, B.L. Transcriptional control of endothelial cell development. Dev. Cell 16, 180–195 (2009).
Birdsey, G.M. et al. Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin. Blood 111, 3498–3506 (2008).
De Val, S. et al. Combinatorial regulation of endothelial gene expression by ETS and forkhead transcription factors. Cell 135, 1053–1064 (2008).
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).
Kataoka, H. et al. Etv2/ER71 induces vascular mesoderm from Flk1+PDGFRα+ primitive mesoderm. Blood 118, 6975–6986 (2011).
Acknowledgements
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
Contributions
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
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
About this article
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). https://doi.org/10.1038/nprot.2015.126
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2015.126
This article is cited by
-
Etv2 regulates enhancer chromatin status to initiate Shh expression in the limb bud
Nature Communications (2022)
-
Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions
Scientific Reports (2021)
-
Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis
Nature (2020)
-
In vitro conversion of adult murine endothelial cells to hematopoietic stem cells
Nature Protocols (2018)
-
Amniotic fluid cells: current progress and emerging challenges in renal regeneration
Pediatric Nephrology (2018)
Comments
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.