ESC-derived VE-cadherin⁺CD45⁻ cells have endothelial properties. Undifferentiated GFP-transfected ESCs (ES) and subsequent differentiating cells were analyzed by FACS. GFP⁺PI⁻ cells were gated as ESC-derived viable cells. (A) Percentage of gated cells among the total cells is specified. (B) Sequential analysis of the percentage of cells positive for each antigen among ESC-derived viable cells. Data are presented as mean ± SD of 3 independent experiments. (C) VE-cadherin⁺CD45⁻(VEcad⁺CD45⁻) or VE-cadherin⁻CD45⁻(VEcad⁻CD45⁻) cells were sorted on day 10. Representative FACS dot plots and percentages of gated cells are shown. Purities of viable VE-cadherin⁺CD45⁻ and VE-cadherin⁻CD45⁻ cells are 99.2% ± 0.6% and 99.9% ± 0.1%, respectively, from at least 3 independent experiments. (D) VE-cadherin⁺ cells on day 10 were analyzed by FACS with various mAbs. Percentages of cells positive for each antigen among VE-cadherin⁺ cells are shown. Gray line indicates isotype control; black line, VE-cadherin⁺ cells. (E-J) Untransfected ESC-derived VE-cadherin⁺CD45⁻ cells sorted on day 10 were evaluated by the DiI-Ac-LDL incorporation assay (E) or immunostaining with IgG1 (F), anti–Hb (G), VEGFR-2 (H), eNOS (I), or VWF (J) Abs. Hoechst 33342 (E-J) and anti–VE-cadherin mAb (F-J) were used to detect nuclei and endothelial cells, respectively. Original magnification × 200. Scale bar, 50μm.