Notch signaling clonally controls the hematopoietic vs endothelial lineage specification of hPSC-derived hemogenic precursors and proposed model for hemato-endothelial lineage specification of hPSCs via HES1. (A) Schematic for the experiment performed to determine the role of Notch signaling in hematopoietic vs endothelial lineage specification of isolated hemogenic precursors at the clonal level. Isolated hemogenic precursors were directly dispensed into 96-well plates and then underwent hemato-endothelial specification in the presence or absence of GSI or Jag1. The specified cells were distinguished by immunocytochemical staining with CD45 and eNOS. The number of wells having hematopoietic, endothelial, or bipotent cells was quantified on a plate reader. (B) Morphologic and phenotypic distinctions among hematopoietic, endothelial, and bipotent cells specified from single hemogenic precursors upon GSI or Jag1 treatment. Each well was counted as hematopoietic (HEM), endothelial (ENDO), or bipotent (Bipotent) cells depending on whether the majority of the cells in the wells were positive for CD45+ (HEM), eNOS+ (ENDO), or CD45+/eNOS+ (Bipotent). Scale bars represent 100 μm. White arrowhead, CD45+ cell; yellow arrowhead, eNOS+ cell. (C-E) Lineage specification of isolated single precursors upon Notch inhibition (GSI, D) or Notch activation (Jag1, E) compared with endogenous developmental potential (Basal, C). Frequencies of wells positive for CD45+ (HEM), eNOS+ (ENDO), and CD45+/eNOS+ (Bipotent) were significantly different in GSI (D) or Jag1 (E) treatments compared with controls. *P < .05; **P < .01 (n = 3). (F) The proposed model suggests that HES1, the downstream effector of Notch signaling, is a key regulator of hemogenic specification and subsequent commitment of hemogenic precursors to hematopoietic or endothelial lineages in hPSCs.