HES1 is a key modulator of commitment to the hematopoietic or endothelial fate during development. (A) HES1 knockdown achieved by siRNA in hemogenic precursors formed fewer hematopoietic colonies (8 ± 2.9 vs 30.3 ± 4.4). *P < .05 (n = 4). (B-C) Retroviral knockdown of HES1 impaired blood production (B, 0.36 ± 0.12 × 103 cells vs 3.85 ± 0.98 × 103 cells) and hematopoietic colony formation (C, 15.5 ± 1.29 vs 25.25 ± 3.59). **P < .01 (n = 4). (D) Colonies formed after HES1 siRNA knockdown represented typical hematopoietic colony subtypes, including granulocytes (CFU-G, yellow arrowhead in left panel), macrophages (CFU-M, green arrowhead in left panel), granulocyte macrophages (CFU-GM, middle panel), and erythrocytes (CFU-E, right panel). Scale bars represent 100 μm. (E) siRNA-mediated HES1 knockdown in hemogenic precursors increased the number of endothelial colonies (12.0 ± 3.2 vs 3.3 ± 2.1) relative to controls. *P < .05 (n = 4). (F-G) Retroviral HES1 suppression induced a higher level of endothelial-specific VE-cadherin expression (F, 74.3 ± 5.89% vs 31.73 ± 3.07%) and total number of endothelial colonies (G, 9.25 ± 1.26 vs 1 ± 1.15) than the control. **P < .01 (n = 4). (H) Characterization of hPSC-derived endothelial cells in HES1 knockdown cultures shown by morphology (bottom left panel), VE-cadherin staining with Dil-Ac-LDL uptake (bottom middle panel), and functional in vitro tube formation (bottom right panel) compared with HUVEC positive controls (upper row). Scale bars represent 100 μm (left and middle panels) and 250 μm (right panels). (I-K) Relative expression of HES1 (I) and hematopoietic markers, Runx1 (J) and TAL1 (K), in hematopoietic and endothelial colonies harvested from CFU assays upon Jag1 and GSI stimulation, respectively. Taken together with HES1 (I, 12.74 ± 3.39-fold), higher expression levels of Runx1 (J, 55.12 ± 8.88-fold) and TAL1 (K, 2.3 ± 0.05-fold) were observed in hematopoietic colonies vs endothelial colonies (1.44 ± 0.24-fold; F, 5.44 ± 1.01-fold; G, 0.3 ± 0.06-fold). HUVEC was used as a control. **P < .01 (n = 3). (L) Quantitative analysis of endothelial-specific gene expression relative to HUVEC demonstrating GSI-mediated Notch knockdown increased endothelial-specific KDR (0.49 ± 0.02-fold vs 0.08 ± 0.02-fold), VE-cadherin (0.46 ± 0.2-fold vs nondetected), and Tie2 (0.48 ± 0.1-fold vs nondetected) gene expression over Jag1 stimulation. Expression level of PECAM1 was similar in both GSI-treated (0.38 ± 0.03-fold) and Jag1-treated (0.31 ± 0.08-fold) cells.
