Norepinephrine Improves The Generation Of Hematopoietic Cells From Human Pluripotent Stem Cells With Increased Functional Properties

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 from the developing peripheral nervous system (PNS) has been implicated in the generation of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos. Our own observations of AGM and urogenital ridge (UR) explants from a 6 weeks old human embryo show neurogenic potential. Since the AGM and UR are active sites of hematopoietic emergence, we hypothesize that migrating neural crest (NC) cells, precursors of the PNS, play an active role in HSC generation and maturation. Given that, at the time of HSC emergence, NC cells start expressing enzymes required for catecholamine production, we added norepinephrine (NE) to our optimized hPSC differentiation system and assessed for hematopoietic progenitor cell output. We observed approximately 50% increase of cells with an HSC immunophenotype (CD43+CD34+CD38-CD90+CD45RA-) compared to control settings (1.56 ± 0.25 fold increase, n=5 independent experiments, p=0.007). We identified this phenotype as an early progenitor cell with lymphoid and myeloid differentiation potential as well as highest colony forming unit (CFU) content. Importantly, the increase was specific to this cell fraction, since the proportion of more mature progenitors (CD43+CD34+ cells) and of total blood (CD43+) did not display a significant increase in the presence of NE (respectively, 1.053 ± 0.09 fold increase p=0.2104, and 0.97 ± 0.46 fold increase p=0.880, n=5 independent experiments). Continuing the differentiation culture for 5 additional days did not show additional increase in the frequency of these early progenitors. This indicates that higher proportion of early progenitors in the presence of NE is due to increased emergence from hemogenic endothelium rather than to maintenance of the early progenitors in culture. Improved generation of early progenitors was also consistent with an increase of colonies in the CFU assay (1.84 ± 0.29 fold increase in colony numbers n=4 independent experiments, p=0.010). The increased output of early hematopoietic progenitors was reversed when the Adrenergic Receptor β2 specific inhibitor ICI 118,551 was added together with NE, showing that the effect of NE is mediated by the activation of this receptor. Our preliminary results of transplantation in NSG mice show CD43+ human chimerism at week 6 post-transplantation 11 fold higher in mice transplanted with hematopoietic cells generated in the presence of NE compared to their counterparts transplanted with cells generated in standard conditions [2.33 ± 1.25% (n=3) vs 0.21 ± 0.02% (n=2), p=0.006]. However, since the detected human cells are predominantly of myeloid lineage (CD33+/CD15+) and the chimerism levels decrease back to levels comparable to PBS transplanted negative control at week 8-10 post-transplantation, this suggests that NE increases the homing and short term repopulation ability of pluripotent stem cell derived early hematopoietic progenitors.