ESC-derived VE-cadherin⁺CD45⁻ cells generate hematopoietic and endothelial cells/colonies. Light micrographs of adherent hematopoietic clusters on days 10 + 5 (A) and 10 + 23 (B). (C-D) May-Giemsa staining of floating hematopoietic cells on days 10 + 6 (C) and 10 + 30 (D). (E) Immunostaining of floating hematopoietic cells on day 10 + 30 with anti–CD41 (red) and Hb (green) Abs. (F-K) Light micrographs and May-Giemsa staining of a GM (F-G), erythroid (H-I), or mixed colony (J-K) are depicted. (L) Immunostaining of an endothelial colony with anti–VE-cadherin mAb (blue). (M) Immunostaining of VE-cadherin⁺CD45⁻ cell-derived cells after 7-day culture with anti–VE-cadherin (red) and VWF (green) Abs. (E, M) Nuclei were labeled with Hoechst 33342. (N, O) Numbers of CD45⁺ (N) or CD45⁺CD34⁺ cells (O) derived from 1 × 10⁵ VE-cadherin⁺CD45⁻ (VEcad⁺CD45⁻) or VE-cadherin⁻CD45⁻ (VEcad⁻CD45⁻) cells. (P) Sequential analysis of the percentages of nucleated erythrocytes (red), enucleated erythrocytes (yellow), myeloid lineage cells (green), and megakaryocytes (blue) among floating cells. Each bar represents the mean of 3 independent experiments. (Q) Sequential analysis of the numbers of hematopoietic colonies per 1 × 10⁴ VEcad⁺CD45⁻ or VEcad⁻CD45⁻ cell-derived cells. (R) Numbers of endothelial colonies per 1 × 10³ VEcad⁺CD45⁻ or VEcad⁻CD45⁻ cells. (A-P) Data obtained from VEcad⁺CD45⁻ cells are shown. Data are presented as mean ± SD of 3 independent experiments in N-O and Q-R. Each experiment was performed in triplicate in P-R. Original magnification × 40 (B, F, L), × 100 (A, H, J), and × 200 (C-E, G, I, K, M). Scale bars, 20 μm (C-E, G, I, K), 50 μm (M), and 100 μm (A-B, F, H, J, L).