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

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.

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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.

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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

Authors

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

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)

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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

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  • DOI: https://doi.org/10.1038/nprot.2015.126

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