α4-Integrin⁺ subpopulation among VE-cadherin⁺CD45⁻ cells generates hematopoietic colonies composed of primitive and definitive erythrocytes. (A-H) Light micrographs and May-Giemsa staining of colonies consisting of primitive (A-B) and definitive (E-F) erythrocytes from VE-cadherin⁺α4-integrin⁺ cells are depicted. Immunostaining data from colonies consisting of primitive (C-D) and definitive (G-H) erythrocytes from VE-cadherin⁺α4-integrin⁺ cells are also presented. Cy3 detection of erythrocytes stained with anti–β-globin (C, G) or anti–ζ-globin (D, H) mAbs (red) and FITC detection with anti-Hb polyclonal Ab (green). Nuclei were labeled with Hoechst 33342. Merged images are shown. Original magnification × 100 (A, E) and × 200 (B-D, F-H). Scale bars, 50 μm. (I-J) Numbers of hematopoietic colonies per 1 × 10⁴ cells sorted on day 10 (I) or after 30-day coculture on OP9 layers (J) in each subpopulation. VEcad⁺ denotes the VE-cadherin⁺CD45⁻ population. EryP and EryD represent colonies consisting of primitive and definitive erythrocytes, respectively. Data are presented as mean ± SD of 3 (I) or 2 (J) independent experiments. Each experiment was performed in duplicate (I) or triplicate (J).