Figure 3.

The CD34⁺CD14⁻ subpopulation among VEC⁺CD31⁺ ECs represents an endothelial progenitor population, and Notch signaling controls their proliferation or further maturation. (A) Schematic of the single-cell culture experiment. On day 6 in the endothelial differentiation, the single cells of the three subpopulations (CD34⁺CD14⁻, CD34⁻CD14⁻, CD14⁺) among hESC-derived VEC⁺CD31⁺ cells were sorted onto 96 well plates (1 cell/well) and cultured under endothelial cell conditions for 14 days. (B, C) Percent of single cells dividing (B), defined as undergoing ≥ 1 cell division, and the average number of cell progeny per well after 14 days (C) were measured in each EC subpopulation. ^*P < 0.0001 vs CD34⁻CD14⁻ and CD14⁺ EC. (D) Representative photomicrographs of the single CD34⁻CD14⁻ (top), CD34⁺CD14⁻ (middle), and CD14⁺ EC (bottom) culture assay. Scale bars, 50 μm (day 0) and 100 μm (day 3, day 9 and day 14). Day 3, day 9 and day 14, brightfield; day 0 and insets of day 14, VEC-EGFP. (E) Expression of CD34 and KDR on day 14 in the cultured cells derived from the EPs (VEC⁺CD31⁺CD34⁺CD14⁻) sorted on day 6. After sorting, EPs were cultured with VEGF-A and analyzed with FACS on day 14. (F) Representative images of western blot analysis for active Notch1 (the Notch1 intracellular domain (N1ICD)) in nuclear extracts of the cultured cells treated with VEGF-A with or without rhDll4 or DAPT. (G) Quantitative results in F are shown. Bands were scanned and quantified with Image J. The signal intensity was normalized to β-actin expression. (−) indicates no molecules. ^*P < 0.05 vs (−), ^**P < 0.01 vs (−) and VEGF-A, #P < 0.01 vs VEGF-A, and VEGF-A + rhDll4. (H) Diverse ratios of VEC⁺CD31⁺CD34⁺CD14⁻ cells among VEC⁺CD31⁺ cells on day 14. 1 × 10⁴ VEC⁺CD31⁺CD34⁺CD14⁻ cells (EPs) were sorted on day 6 and cultured with VEGF-A with or without rhDll4 or DAPT in phase 3. Notably, combined treatment with VEGF-A and DAPT maintained a higher ratio of VEC⁺CD31⁺CD34⁺CD14⁻ cells among the cultured cells on day 14, whereas VEGF-A and rhDll4 decreased that ratio (P < 0.01). (I) The differential EC (VEC⁺CD31⁺) and EP (CD34⁺CD14⁻ EC) ratios on day 6 induced with treatment with VEGF-A with or without rhDll4 or DAPT in phase 2. Error bars, SD (n ≥ 3).