Title | In vitro conversion of adult murine endothelial cells to hematopoietic stem cells. |
Publication Type | Journal Article |
Year of Publication | 2018 |
Authors | Durán JGabriel Ba, Lis R, Lu TM, Rafii S |
Journal | Nat Protoc |
Volume | 13 |
Issue | 12 |
Pagination | 2758-2780 |
Date Published | 2018 Dec |
ISSN | 1750-2799 |
Keywords | Animals, 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> |
DOI | 10.1038/s41596-018-0060-3 |
Alternate Journal | Nat Protoc |
PubMed ID | 30429596 |
PubMed Central ID | PMC9923715 |
Grant List | R01 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 |