Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification.
Moon:Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.
Asterisk with author names denotes non-ASH members.