PLX123144

GSE88840: Conversion of adult endothelial cells into immune-competent long-term repopulating authentic haematopoietic stem cells

• Organsim mouse
• Type RNASEQ
• Target gene
• Project ARCHS4

The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into true engraftable haematopoietic stem cells (HSCs) remain undefined. This is due to lack of models to recreate the ephemeral transition from endothelial to haemogenic cells and to HSCs. Here, we have developed a novel sequential in vitro model in which by precise conditional sequential on-off expression of transcription factors: FosB, Gfi1, Runx1, and Spi1 (FGRS); and re-establishing a proper inductive vascular niche, we were able to reprogram adult mouse ECs into HSCs (rEC-HSCs) over a 28 day period. During the day 0-8 induction phase, FGRS are conditionally expressed in adult non-lymphatic ECs isolated from Runx1-IRES-GFP reporter mice and co-cultured with vascular niche. At specification phase (day 8-20), FGRS-transduced VEcad+Runx1-CD45- ECs switch on the endogenous expression of Runx1, initiating haematopoietic program and silencing EC fate in VEcad+Runx1+CD45+ cells, setting the stage for full commitment to VEcad-Runx1+CD45+ haematopoietic stem and progenitor cells (rEC-HSPCs). During expansion phase (day 20-28), the niche cells promote expansion of a large number of haematopoietic cells, at which time expression of exogenous FGRS is turned off. Subsets of rEC-HSPCs are endowed with distinctive features of rEC-HSCs, including single cell clonal multi-lineage reconstituting potential, ability to serially re-establish bone marrow HSPC compartments, reconstitute adaptive immune function in Rag1-/- mice, and long-term self-renewal capacity. Employing Runx1-IRES-GFP reporter mice enabled deconvolution of stage-specific pathways involved in generation of engraftable rEC-HSCs. Inhibition of TGF signaling along with activation of BMP and CXCL12 pathways reinforced the induction phase. Cxcr4 deficient ECs failed to undergo conversion to rEC-HSPCs, indicating that CXCL12 signaling is essential for specification and expansion phases. This reprogramming platform provides an interrogable platform to decipher pathways involved in the transition of ECs into haematopoietic cells. Our approach also facilitates devising strategies to reprogram adult ECs into abundant autologous HSCs amenable to genetic modification for treatment of genetic and acquired haematological disorders. SOURCE: Balvir Kunar, Jr. (bak2010@med.cornell.edu) - Shahin Rafii, M.D. Weill Cornell Medical College