Fig. 1. Characterization of the flk-1⁺ and PDGFR⁺ EB cells generated with BMP4 in the serum-free medium. (A) Generation of the flk-1⁺ and PDGFR⁺ EB cells. The undifferentiated E14 ES cells (a) and the cells from EBs cultured for 4.6 days in the BMP4-containing serum-free medium (b) were stained with Flk-1-PE and PDGFR-bio. The latter group was further stained with SA-APC. Single positive cells, such as flk-1^–PDGFR⁺ cells (R3) and flk-1⁺PDGFR^– cells (R4), as well as DP cells (R5) and DN cells (R6), were then analyzed (a) and sorted (b) with Vantage SE. Viable cell populations were pre-enriched by propidium iodide exclusion (R1) as described previously (Nakayama et al., 2000). For sorting, stained EB cells were pre-gated with R2 to further reduce dead cell contamination (b, left). R6 was determined by the corresponding isotype control staining (a, middle; b, middle). Populations of R3 to R5, indicated as % total EB cells, are: (a) R3: 0.7, R4: 0.0, R5: 0.3 (right) and as for the isotype control, R3: 0.7, R4: 1.3, R5: 0.5 (middle); (b) R3: 26.4, R4: 7.7, R5: 9.2 (right) and as for the isotype control, R3: 0.5, R4: 0.0, R5: 0.2 (middle). These results are representative of 17 independent experiments. (B) Erythro-myeloid CFCs in the flk-1⁺ and PDGFR⁺ EB cell fractions, with or without culturing on the OP9 cells. Individual FACS-purified cell fractions were either directly plated in the serum-free methylcellulose medium (–) or cultured for 3 days on OP9 (+). The latter cells were then plated into the same medium for hematopoietic colony formation. Total CFC numbers were averaged over four to six independent experiments and are shown with the corresponding s.d. (vertical line). R3: flk-1^–PDGFR⁺, R4: flk-1⁺PDGFR^–, R5: DP, R6: DN. (C) Lymphoid potentials of the flk-1⁺ and PDGFR⁺ EB cells. Individual FACS-purified cell fractions were cultured for 12 days on OP9 in the presence of IL-2 and IL-7. The entire culture was then harvested, stained with B220-PE and Sca-1-FITC and analyzed by FACScan. Populations of B220⁺Sca-1^–, B220^–Sca-1⁺, and B220⁺Sca-1⁺ cells are indicated as % of total cells. The scatter patterns, the gate settings and the isotype control staining patterns (IgG-FITC/IgG-PE) were essentially the same as described previously (Nakayama et al., 1998). Note that the OP9 cells were B220^–Sca-1^–. Cellularity, reflected by the density of the plot, was highest in (a) and (c), owing to the massive accumulation of lymphocytic cells. In contrast, small numbers of lymphocytic cell foci were observed in d, and no such cells were detected in b and e. These results are representative of six independent experiments. (a), flk-1⁺PDGFR^– (isotype control staining for c); (b), flk-1^–PDGFR⁺; (c), flk-1⁺PDGFR^–; (d), DP; (e), DN.