Regulation of hPSC hemogenic precursor specification by Notch signaling. (A-B) Effect of Jag1-mediated Notch activation on hemogenic precursor specification of hESCs. Increased frequency (A, 63.1 ± 15.6% vs 29.6 ± 4.5%) and total number (B, 11 ± 3.2 × 10⁴ cells vs 4.4 ± 0.4 × 10⁴ cells) of hemogenic precursors were observed in hEBs treated with Jag1 relative to controls on day 10. In contrast, on day 15, the frequency (A, 29.7 ± 10.8% vs 55.1 ± 10.6%) and total number (B, 5.3 ± 2.5 × 10⁴ cells vs 9.1 ± 1.2 × 10⁴ cells) of hemogenic precursors in Jag1-treated hEBs had significantly decreased. *P < .05 (n = 3). (C-D) Effect of GSI on the emergence of hemogenic precursors during hEB development. hEBs treated with a GSI showed decreased frequency (C, 1.58 ± 1.36% vs 7.08 ± 1.08%) and total number (D, 5.2 ± 4.1 × 10³ cells vs 29.6 ± 8.0 × 10³ cells) of hemogenic precursor relative to controls on day 10. *P < .05 (n = 3). (E) GSI inhibited hemogenic precursor augmentation from hEBs formed with hFib-iPSCs (24.3 ± 1.1 × 10³ cells vs 39.6 ± 1.2 × 10³ cells). *P < .05 (n = 3). (F-I) Effect of Notch signaling on the emergence of hemogenic precursors from hESCs was confirmed by colocalization of PECAM1 with Notch1 (F-G) and HES1 (H-I) by immunofluorescence. Day 10 hEBs stimulated by Jag1 (G,I) were compared with control hEBs (F,H). In EBs stimulated with Jag1, the majority of Notch1+ cells (G) and HES1+ cells (I) coexpressed hemogenic precursor marker PECAM1 (white arrowhead), whereas lower numbers of PECAM1+ cells (red arrowheads) are predominant in control hEBs. Scale bars represent 100 μm. (J) HES1 knockdown during hematopoietic hEB development impaired hemogenic precursor specification. HES1 siRNA–treated hEBs exhibited reduced numbers of hemogenic precursors compared with the scrambled siRNA control (27.6 ± 7.4 × 10³ cells vs 52.1 ± 7.1 × 10³ cells). *P < .05 (n = 3).