Norepinephrine improves the generation of hematopoietic cells from human pluripotent stem cells

The possibility of differentiating human pluripotent stem cells (hPSCs) to hematopoietic stem cells (HSCs) could provide an unlimited source of donor cells for the treatment of hematological disorders and malignancies where HSC transplantation is required. Recently, signaling fromthe developingperipheral nervous system(PNS)has been implicated in the generation of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos. Our own observations of explants of AGM and urogenital ridge (UR) explants from6weeksold human embryos showneurogenic potential. Since theAGMandURare active sites of hematopoietic emergence,we hypothesize thatmigrating neural crest (NC) cells, precursors of the PNS, might have an active role in HSC generation. Given that, at the time of HSC emergence, NC cells express enzymes required for catecholamine production, we added norepinephrine (NE) to our optimized hPSCdifferentiation system and assessed for hematopoietic progenitor cell output. We observed a 2 fold increase of cells with an HSC phenotype (CD43+CD34+CD38-CD90+CD45RA-) compared to control settings.We identified this phenotype as a cellwithhighest lymphoid andmyeloid differentiation potential. Importantly, the increase was specific to this most immature fraction, since the proportion of progenitors (CD43+CD34+CD38cells) and of total blood (CD43+) did not display a significant increase in the presence of NE. Continuing the differentiation culture for 5 additional days did not show additional increase in the number of HSCs. This indicates that higher proportion of HSCs in the presence of NE is due to increased emergence from hemogenic endothelium rather than to better maintenance of the cells in culture. Improved generation of immature progenitors was also consistent with an increase of colonies in the colony forming unit assay. The increased output of primitive hematopoietc progenitors was reversed when the Adrenergic Receptor b2 specific inhibitor ICI 118,551 was added together with NE, showing that the effect of NE is mediated by the activation of this receptor. Currently we are in the process of transplanting these cells in immunocompromised mice, in order to determine whether NE is the factor needed to achieve engraftment of hPSC-derived HSCs.