Hartman Institute for Therapeutic Organ Regeneration

In vitro conversion of adult murine endothelial cells to hematopoietic stem cells.

TitleIn vitro conversion of adult murine endothelial cells to hematopoietic stem cells.
Publication TypeJournal Article
Year of Publication2018
AuthorsDurán JGabriel Ba, Lis R, Lu TM, Rafii S
JournalNat Protoc
Volume13
Issue12
Pagination2758-2780
Date Published2018 Dec
ISSN1750-2799
KeywordsAnimals, Cell Culture Techniques, Cell Differentiation, Cell Proliferation, Cells, Cultured, Cellular Reprogramming Techniques, Endothelial Cells, Gene Expression Regulation, Hematopoietic Stem Cells, Human Umbilical Vein Endothelial Cells, Humans, Mice, Transcription Factors, Transcriptome
Abstract

<p>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.</p>

DOI10.1038/s41596-018-0060-3
Alternate JournalNat Protoc
PubMed ID30429596
PubMed Central IDPMC9923715
Grant ListR01 HL115128 / HL / NHLBI NIH HHS / United States
R35 HL150809 / HL / NHLBI NIH HHS / United States
RC2 DK114777 / DK / NIDDK NIH HHS / United States
R01 HL139056 / HL / NHLBI NIH HHS / United States
R01 HL119872 / HL / NHLBI NIH HHS / United States
U01 AI138329 / AI / NIAID NIH HHS / United States
R01 HL128158 / HL / NHLBI NIH HHS / United States
R01 DK095039 / DK / NIDDK NIH HHS / United States

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Hartman Institute for Therapeutic Organ Regeneration
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