Assessment of Endothelial-to-Hematopoietic Transition of Individual Hemogenic Endothelium and Bulk Populations in Defined Conditions.

Endothelial-to-hematopoietic transition (EHT) is a unique morphogenic event in which flat, adherent hemogenic endothelial (HE) cells acquire round, non-adherent blood cell morphology. Investigating the mechanisms of EHT is critical for understanding the development of hematopoietic stem cells (HSCs) and the entirety of the adult immune system, and advancing technologies for manufacturing blood cells from human pluripotent stem cells (hPSCs). Here we describe a protocol to (a) generate and isolate subsets of HE from hPSCs, (b) assess EHT and hematopoietic potential of HE subsets in bulk cultures and at the single-cell level, and (c) evaluate the role of NOTCH signaling during HE specification and EHT. The generation of HE from hPSCs and EHT bulk cultures are performed in xenogen- and feeder-free system, providing the unique advantage of being able to investigate the role of individual signaling factors during EHT and the definitive lympho-myeloid cell specification from hPSCs.