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Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

Nature (2017-05-18)
Raphael Lis, Charles C Karrasch, Michael G Poulos, Balvir Kunar, David Redmond, Jose G Barcia Duran, Chaitanya R Badwe, William Schachterle, Michael Ginsberg, Jenny Xiang, Arash Rafii Tabrizi, Koji Shido, Zev Rosenwaks, Olivier Elemento, Nancy A Speck, Jason M Butler, Joseph M Scandura, Shahin Rafii
ABSTRACT

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

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Heparin sodium salt from porcine intestinal mucosa, Grade I-A, ≥180 USP units/mg
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PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Distributed for Phanos Technologies
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Medium 199, With Earle′s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
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Dulbecco′s Modified Eagle′s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium bicarbonate, without L-glutamine, liquid, sterile-filtered, suitable for cell culture
Sigma-Aldrich
PKH67 Green Fluorescent Cell Linker Kit for General Cell Membrane Labeling, Distributed for Phanos Technologies