Hematopoietic progenitors exclusively reside in the α4-integrin⁺ subpopulation among VE-cadherin⁺CD45⁻cells. (A) VE-cadherin⁺α4-integrin⁻ (VEcad⁺α4⁻), VE-cadherin⁺α4-integrin⁺ (VEcad⁺α4⁺), or VE-cadherin⁻α4-integrin⁺ (VEcad⁻α4⁺) cells were sorted on day 10 after the exclusion of PI⁺ and CD45⁺ cells. Representative FACS dot plots and percentages of gated cells are shown. Purities of viable VEcad⁺α4⁻, VEcad⁺α4⁺, and VEcad⁻α4⁺ cells are 95.0%± 3.0%, 94.1%± 0.5%, and 96.9%± 2.1%, respectively, from at least 3 independent experiments. (B-D) Light micrographs of adherent hematopoietic clusters on day 10 + 6 from VEcad⁻α4⁺ cells (B) and days 10 + 6 (C) and 10 + 18 (D) from VEcad⁺α4⁺ cells. (E-G) May-Giemsa staining of floating hematopoietic cells on day 10 + 6 from VEcad⁻α4⁺ cells (E) and days 10 + 6 (F) and 10 + 30 (G) from VEcad⁺α4⁺ cells. Original magnification × 40 (B-D) and × 200 (E-G). Scale bars, 100 μm (B-D) and 20 μm (E-G). (H) Sequential analysis of the numbers of floating viable cells per cultured 1 × 10⁴ VE-cadherin⁺ (▪), VE-cadherin⁻ (□), VEcad⁺α4⁻ (○), VEcad⁺α4⁺ (•), or VEcad⁻α4⁺ (▴) cells. (I) Numbers of endothelial colonies per 1 × 10³ cells in each subpopulation. (H-I) Data are presented as mean ± SD of 3 independent experiments. Each experiment was performed in triplicate. (J) RT-PCR analysis of genes associated with hematopoietic or endothelial development. Each lane contained cDNA from the following cells: adult cynomolgus monkey BM cells (lane 1), K562 erythroblastic cells (lane 2), human umbilical vein endothelial cells (lane 3), OP9 stromal cells (lane 4), total GFP⁺ ESC-derived cells (lane 5), VE-cadherin⁺α4-integrin⁻ cells (lane 6), VE-cadherin⁺α4-integrin⁺ cells (lane 7), and VE-cadherin⁻α4-integrin⁺ cells (lane 8) sorted on day 10. Representative results from 1 of 3 independent experiments are shown.