Modulation of WNT, Activin/Nodal, and MAPK Signaling Pathways Increases Arterial Hemogenic Endothelium and Hematopoietic Stem/Progenitor Cell Formation During Human iPSC Differentiation.

Several differentiation protocols enable the emergence of hematopoietic stem and progenitor cells (HSPCs) from human-induced pluripotent stem cells (iPSCs), yet optimized schemes to promote the development of HSPCs with self-renewal, multilineage differentiation, and engraftment potential are lacking. To improve human iPSC differentiation methods, we modulated WNT, Activin/Nodal, and MAPK signaling pathways by stage-specific addition of small-molecule regulators CHIR99021, SB431542, and LY294002, respectively, and measured the impact on hematoendothelial formation in culture. Manipulation of these pathways provided a synergy sufficient to enhance formation of arterial hemogenic endothelium (HE) relative to control culture conditions. Importantly, this approach significantly increased production of human HSPCs with self-renewal and multilineage differentiation properties, as well as phenotypic and molecular evidence of progressive maturation in culture. Together, these findings provide a stepwise improvement in human iPSC differentiation protocols and offer a framework for manipulating intrinsic cellular cues to enable de novo generation of human HSPCs with functionality in vivo.