One of the most promising objectives of clinical hematology is to derive engraftable autologous hematopoietic stem cells (HSCs) from human induced pluripotent stem cells (iPSCs). Progress in translating iPSC technologies to the clinic relies on the availability of scalable differentiation methodologies. In this study, human iPSCs were differentiated for 21 days using STEMdiff™, a monolayer-based approach for hematopoietic differentiation of human iPSCs that requires no replating, co-culture or embryoid body formation. Both monolayer and suspension cells were functionally characterized throughout differentiation. In the supernatant fraction, an early transient population of primitive CD235a+ erythroid cells first emerged, followed by hematopoietic progenitors with multilineage differentiation activity in vitro but no long-term engraftment potential in vivo. In later stages of differentiation, a nearly exclusive production of definitive erythroid progenitors was observed. In the adherent monolayer, we identified a prevalent population of mesenchymal stromal cells and limited arterial vascular endothelium (VE), suggesting that the cellular constitution of the monolayer may be inadequate to support the generation of HSCs with durable repopulating potential. Quantitative modulation of WNT/β-catenin and activin/nodal/TGFβ signaling pathways with CHIR/SB molecules during differentiation enhanced formation of arterial VE, definitive multilineage and erythroid progenitors, but was insufficient to orchestrate the generation of engrafting HSCs. Overall, STEMdiff™ provides a clinically-relevant and readily adaptable platform for the generation of erythroid and multilineage hematopoietic progenitors from human pluripotent stem cells.HighlightsRobust, scalable and clinically-relevant monolayer-based culture system for hematopoietic differentiation of human iPSCs.Successive emergence of primitive erythroid cells, definitive multilineage HSPCs and erythroid progenitors in the culture supernatant.Abundant mesenchymal cells and limited arterial vascular endothelium in the culture monolayer.CHIR/SB molecules increase arterial vascular endothelium formation, suppress primitive hematopoiesis and promote definitive multilineage and erythroid progenitors.

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