Direct Conversion of Adult Endothelial Cells into Immunecompetent Long-Term Engraftable Clinically Scalable Hematopoietic Stem Cells: Pathway to Therapeutic Translation

• Published in: Blood 2016-12, Vol.128 (22), p.372-372
• Main Authors: Lis, Raphael, Harrasch, Charles, Poulos, Michael Gustave, Duran, Jose Gabriel, Schachterle, William, Ginsberg, Michael, Rafii, Arash, Shido, Koji, Speck, Nancy A., Butler, Jason M., Scandura, Joseph M., Rafii, Shahin
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.372.372

▪ The molecular pathways and microenvironmental cues that choreograph the conversion of endothelial cells (ECs) into true engraftable hematopoietic stem cells (HSCs) remain undefined. This is due to lack of models to recreate the short-lived transition from EC to hemogenic cells and to HSCs. Extending on our previous work (Sandler V. et al, Reprogramming of human endothelium into hematopoietic cells requires vascular niche induction. Nature, 511:312-8. 2014), we have developed a novel, sequential, clinically-translatable in vitro model of the adult EC to hematopoietic transition (EHT). This model uses precise, conditional, on-off expression of transcription factors (FosB, Gfi1, Runx1, and Spi1 - FGRS) and an inductive vascular niche to reprogram adult mouse ECs into true HSCs (rEC-HSCs) with high efficiency. During the induction phase (days 0-8), FGRS are conditionally expressed in adult non-lymphatic ECs isolated from Runx1-IRES-GFP reporter mice and co-cultured with the supportive vascular niche cells. During the specification phase (days 8-20), FGRS-transduced VEcad+Runx1-CD45- ECs activate expression of endogenous Runx1, initiating the hematopoietic program and silencing EC fate. The VEcad+Runx1+CD45+ cells, then complete specification and full commitment to VEcad-Runx1+CD45+ hematopoietic stem and progenitor cells (rEC-HSPCs). Specified rEC-HSPCs are then expanded (days 20-28) on the vascular niche generating a large number of hematopoietic cells and, at this point, expression of exogenous FGRS is turned off. Transplantation of rEC-HSPCs (CD45.2) into lethally irradiated (CD45.1) recipient mice reconstitute both short-term and long-term hematopoiesis, and are capable of engrafting secondary and tertiary recipients (rEC-HSCs). Once engrafted, rEC-HSPCs give rise to functional myeloid and lymphoid cells with full complement of polarized T cell subsets. rEC-HSC-derived immune cells undergo T-cell receptor (TCR) rearrangement and reconstitute adaptive immune function in Rag1-/- mice. To prove the stem cell potential of rEC-HSCs, we performed clonal analyses on the VEcad+Runx1+CD45+ cells (days 8-20), in which single cells were plated in coculture with vascular niche. Notably, 7 out of 386 CD45.2+ clones gave rise to expanding colonies that were capable of 4 months primary multilineage engraftment into lethally irradiated CD45.1+ recipients. In addition, limiting dilution transplantation of isochronic VEcad-Runx1+CD45+ cells indicated that 1 out of 538 rEC