Effect of Notch signaling on the hematopoietic commitment of purified hemogenic precursors. (A-B) GSI-mediated inhibition of Notch signaling in dissociated single cells from unsorted total EBs resulted in an 89.3% reduction of CD45+ blood cell frequency (A) and immunocytochemical staining for CD45 on single cells after extended hematopoietic culture (Hem-culture) revealed lower numbers of CD45+ cells in the GSI-treated group (B). *P < .05 (n = 3). (C) Schematic summary to examine the effect of up- or downregulated Notch signaling on blood commitment of isolated hemogenic precursors. (D) Emergence of CD45+ blood cells could be modulated by controlling Notch signaling in isolated hemogenic precursors. GSI-mediated Notch downregulation inhibited blood commitment (5.38 ± 1.45%) over controls (28.2 ± 12%). Conversely, Jag1-stimulated hemogenic precursors enhanced blood augmentation (78.2 ± 18%). *P < .05; **P < .01 (n = 3). (E) GSI-mediated inhibition of Notch signaling on isolated hemogenic precursors resulted in a significant reduction of CD45+ blood cells by immunocytochemical staining. (F-G) Total number of hematopoietic colonies (F) and endothelial colonies (G) formed from isolated hemogenic precursors by modulating Notch signaling during extended Hem-culture. GSI-mediated Notch downregulation decreased the number of hematopoietic colonies (F, 4.66 ± 2.51), whereas it increased the number of endothelial colonies (G, 7.33 ± 1.52). On the contrary, upregulated Notch upon Jag1 treatment increased the number of hematopoietic colonies (F, 26.67 ± 4.73) but decreased endothelial colonies (G, 1.33 ± 0.58) compared with the number of hematopoietic (F, 14.67 ± 2.52) and endothelial colonies (G, 3 ± 1) in the control. *P < .05; **P < .01 (n = 3). (H) Effect of up- or downregulation of Notch on endothelial-specific marker expression was analyzed by flow cytometry. Expression level of PECAM1 and VE-cadherin was evaluated in isolated hemogenic precursors with activated or suppressed Notch signaling by Jag1 or GSI treatment, respectively. GSI-treated hemogenic precursors showed higher frequency of PECAM1+VE-cadherin+ (54.73 ± 6.17%) over controls (27.94 ± 7.59%) and Jag1 treatment (9.94 ± 3.82%). *P < .05; **P < .01 (n = 3). (I) Effect of Jag1 on blood commitment of isolated hemogenic precursors. Total CD45+ blood cells increased by 3.97-fold relative to controls following extended Hem-culture upon Jag1 stimulation. *P < .05 (n = 3). (J-K) Reduction of CD45+ blood commitment from isolated hemogenic precursors after HES1 knockdown relative to scrambled siRNA control; frequency (J, 64.9% reduction) and total blood cells (K, 82.6% reduction). *P < .05 (n = 3).