AGM-Derived Endothelial Cells and Notch Ligands Provide Embryonic Hematopoietic Stem Cell-Supportive Niches In Vitro

Greater knowledge of embryonic niche signals regulating the establishment, maintenance, and expansion of hematopoietic stem cells (HSC) during development will be essential in deriving therapeutically useful HSC from pluripotent stem cells (PSC). To this end, we have used the murine embryo model to dissect components of embryonic hematopoietic microenvironments which are sufficient to support nascent HSC and their precursors in vitro. We demonstrate that Akt pathway-activated endothelial cells (ECs) derived from the AGM (aorta-gonad-mesonephros) region, a critical site of HSC emergence during development, can substantially increase short and long-term multilineage engraftment potential from isolated embryonic day 11 (E11) VE-Cadherin+/CD45+ AGM-derived hematopoietic cells by co-culture in vitro. Furthermore, preliminary experiments show that co-culture with AGM-ECs also promotes high level, multilineage engraftment capacity from VE-cadherin+/c-kit+ precursors isolated from younger embryos (E9-E10). These results suggest that endothelial cells from an embryonic HSC-producing niche provide signals sufficient to promote maturation of HSC from embryonic precursors and subsequently support early HSC expansion in vitro. Further dissection of required signals for embryonic HSC expansion identified a unique combination of Notch activation by immobilized Notch ligands, cytokines, and small molecule inhibition of the TGF-β pathway, which is sufficient to inhibit differentiation and enhance self-renewal of embryonic, definitive-stage hematopoietic precursors in vitro. Notably, these conditions significantly increased short and long-term, multilineage repopulating HSC from E11 VE-Cadherin+/CD45+, but not E9-10 VE-Cadherin+/c-kit+ AGM-derived hematopoietic cells, indicating AGM-ECs provide additional, yet to be identified, signals for HSC maturation from developmental precursors. These findings have important implications for dissecting critical niche signals for HSC formation and expansion that will be essential for addressing the elusive goal of deriving HSC from pluripotent precursors.