Abstract
The ability to generate hematopoietic stem cells (HSCs) in vitro would have an immeasurable impact on many areas of clinical practice, including trauma, cancer, and congenital disease. In this protocol, we describe a stepwise approach that converts adult murine endothelial cells (ECs) to HSCs, termed ‘reprogrammed ECs into hematopoietic stem and progenitor cells’ (rEC-HSPCs). The conversion, which is achieved without cells transitioning through a pluripotent state, comprises three phases: induction, specification, and expansion. Adult ECs are first isolated from Runx1-IRES-GFP; Rosa26-rtTa mice and maintained in culture under EC growth factor stimulation and Tgfβ inhibition. In the first (induction) phase of conversion (days 0–8), four transcription factors (TFs)—FosB, Gfi1, Runx1, and Spi1 (FGRS)—are expressed transiently, which results in endogenous Runx1 expression. During the second (specification) phase (days 8–20), endogenous Runx1+ FGRS-transduced ECs commit to a hematopoietic fate and no longer require exogenous FGRS expression. Finally, the vascular niche drives robust proliferation of rEC-HSPCs during the expansion phase (days 20–28). The resulting converted cells possess a transcriptomic signature and long-term self-renewal capacity indistinguishable from those of adult HSCs. In this protocol, we also describe functional in vitro and in vivo assays that can be used to demonstrate that rEC-HSPCs are competent for clonal engraftment and possess multi-lineage reconstitution potential, including antigen-dependent adaptive immune function. This approach thus provides a tractable strategy for interrogating the generation of engraftable hematopoietic cells, advancing the mechanistic understanding of hematopoietic development and HSC self-renewal.
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Acknowledgements
We thank J. Downing (St. Jude Hospital) for help in providing materials and for intellectual input. This work was supported by the Ansary Stem Cell Institute, the Starr Foundation Tri-Institutional Stem Cell Initiative Stem Cell Derivation Laboratory Core (Tri-SCI), and grants from the Daedalus Fund for Innovation of Weill Cornell Medicine (Daedalus); the Empire State Stem Cell Board of the New York State Department of Health (NYSTEM C029156); the US National Institutes of Health (NIH); the National Heart, Lung, and Blood Institute (NHLBI; R01HL128158, R01HL139056-01A1, and U01AI138329); the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK RC2DK114777); and the Qatar National Priorities Research Program (NPRP; 8-1898-3-392). J.G.B.D. was supported by the Ansary Stem Cell Institute and NIH/NHLBI R01HL139056-01A1. R.L. was supported by the Ansary Stem Cell Institute, Tri-SCI, NYSTEM C029156, NIH/NHLBI R01HL139056-01A1, NPRP 8-1898-3-392, and a NYSCF Druckenmiller Fellowship D-F56. T.M.L. was supported by Tri-SCI and the Avalon Fund. S.R. was supported by the Ansary Stem Cell Institute, Tri-SCI, Daedalus, NYSTEM C029156, NIH/NHLBI (R01HL128158, R01HL139056-01A1, and U01AI138329), NIH/NIDDK RC2DK114777, and NPRP 8-1898-3-392.
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S.R. envisioned the original idea. J.G.B.D. and R.L. developed the protocol and wrote the manuscript. J.G.B.D. performed the majority of the experiments. T.M.L. contributed to these experiments. R.L. conceived the project and interpreted the data. All authors commented on the manuscript.
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S.R. is the founder of and a nonpaid consultant to Angiocrine Bioscience, New York, NY, USA. The remaining authors declare no competing interests.
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Key references using this protocol
Lis, R. et al. Nature 545, 439–445 (2017): https://doi.org/10.1038/nature22326
Sandler, V. M. et al. Nature 511, 312–318 (2014): https://doi.org/10.1038/nature13547
Seandel, M. et al. Proc. Natl Acad. Sci. USA 105, 19288–19293 (2008): https://doi.org/10.1073/pnas.0805980105
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Barcia Durán, J.G., Lis, R., Lu, T.M. et al. In vitro conversion of adult murine endothelial cells to hematopoietic stem cells. Nat Protoc 13, 2758–2780 (2018). https://doi.org/10.1038/s41596-018-0060-3
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DOI: https://doi.org/10.1038/s41596-018-0060-3
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