VEGF-A₁₆₅ increases the erythroid colony formation resulting from EB treatment with cytokines+BMP-4. (A-D) Following 15 days of differentiation with cytokines+BMP-4 (-VEGF) or cytokines+BMP-4+VEGF-A₁₆₅ (+VEGF), EBs were dissociated and subjected to erythroid-myeloid CFU assays by plating 1 × 10⁵ EB cells in methylcellulose, as described in “Materials and methods.” (A) The mean numbers of erythroid colonies are plotted per 1 × 10⁵ EB cells according to the indicated concentrations of VEGF-A₁₆₅, added at the initiation of EB differentiation (n = 6 experiments for each hESC line). Total numbers of cells recovered from dissociated EBs at day 15 after treatment with or without VEGF-A₁₆₅ were determined by trypan blue exclusion (inset; n = 11). (B) The percentage of the erythroid subtype among all CFU subtypes is shown for the indicated EB treatments (n = 6 for each treatment and each hESC line). (C) The mean numbers of myeloid (left) and erythroid (right) colonies generated after EB treatment with (+VEGF) or without (-VEGF) VEGF-A₁₆₅ are shown (n = 11). (D) The mean numbers of erythroid colonies are plotted according to the indicated EB treatments. VEGF-A₁₆₅ was added (+) or not (-) throughout the entire EB differentiation period, either at the initiation (days 0-15) or at day 10 (days 10-15). When stated, a neutralizing anti—VEGF-A₁₆₅ mAb was also added (+) or not (-) at the initiation of EB (n = 4 experiments for each treatment). For A, C, and D, SDs are represented by vertical bars. ^*P < .05.