Norepinephrine improves de novo emergence of hematopoietic cells in human pluripotent stem cell differentiation cultures

Abstract

In vitro differentiation of human pluripotent stem cells (hPSCs) to hematopoietic stem cells (HSCs) could provide an unlimited source of cells for HSC transplantation for hematological disorders and malignancies. Catecholamine signaling from the peripheral nervous system has recently been implicated in the emergence of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos, and our own observations of AGM and urogenital ridge explants from a 6 weeks old human embryo show neurogenic potential. We thus added norepinephrine (NE) to our hPSC differentiation system and assessed hematopoietic output. We observed 2.5-fold (n=14, 0.01) increase of cells with an HSC-like phenotype (CD43+CD34+CD38-CD90+CD45RA-) in the viable fraction compared to controls. We identified this phenotype as an early progenitor cell with lymphoid and myeloid differentiation potential as well as highest CFU content. Notably, the increase was highest in this fraction compared to more mature progenitors (CD43+CD34+cells) and total blood (CD43+), and was consistent with a 1.5-fold increase of colonies in CFU assays (n=9, p<0.01). Continuing the differentiation culture for 5 more days did not show further increase in the frequency of these early progenitors, indicating that the higher proportion of early progenitors in the presence of NE is due to increased blood emergence rather than to maintenance of these progenitors in culture. Indeed, when adding NE we observed nearly 5-fold (p= 0.05, n=14) increase in the cell fraction with the surface markers of hemogenic precursors of definitive hematopoiesis CD43-CD34highCD90high (Kennedy et al., 2012) compared to controls. As the first emerging hematopoietic cells in mouse express the β2 adrenergic receptor (Adrb2) subtype, we determined the effect of an Adrb2-specific antagonist on the NE responses. We observed that both the HSC phenotype and the CD43-CD34highCD90high fractions remained increased in the presence of the antagonist, suggesting that another adrenergic receptor subtype mediates the effect of NE in the hematopoietic emergence.