Hematopoietic cell colony formation by c-myb knockout endothelial cells is restored by c-Myb induction. (A) VE-cadherin⁺CD31⁺ cells were sorted from differentiating wild-type ES cells and c-mybTet/KO ES cells as described in Figure 2. Cells were recultured (3000 cells/well) for 6 days with OP9 cells and cytokines (SCF, IL3, Epo) in the presence (c-Myb–) or absence (c-Myb+) of Tet (1 μg/mL). To quantify hematopoietic cell colonies, the cultures were overlaid with a semisolid medium from the second day on. Ery indicates erythroid colony; G, granulocyte colony; GM, granulocyte/macrophage colony; M, macrophage colony; GEM, granulocyte/erythrocyte/macrophage colony; GEMMeg, granulocyte/erythrocyte/macrophage/megakaryocyte colony; GMMeg, granulocyte/macrophage/megakaryocyte colony; Meg, megakaryocyte colony. Mean ± SEM from 3 independent experiments are shown (*P < .05). (B) Phase-contrast microscopy images of representative megakaryocyte and GM colonies derived from wild-type (i) and c-Myb–rescued c-myb^–/– endothelial cells (iii) are shown (Nikon Eclipse TE300). Rectangular areas indicated by dashed lines were further magnified (ii,iv). A fluorescence microscopy image (v) of the same megakaryocyte colony (iii) is shown. The colony was picked and stained with May-Grunwald Giemsa solution (vi) (Leica DMLS). Meg indicates megakaryocyte; P, proplatelet; GM, granulocyte/macrophage. Original magnifications: panels i, iii, and v, 100× (Plan Fluor 10×/0.30 NA objective); panel vi, 1000× (C Plan 100×/1.25 NA oil objective).