VEGF-A₁₆₅ requires the presence of additional factors to optimally promote erythroid colony formation. EBs were differentiated for 15 days with the indicated treatments and plated in CFU assays as described in Figure 1. (A-B) Mean numbers of erythroid colonies derived from EBs differentiated with the indicated treatments, with the corresponding SD (vertical bars) (n = 3 independent experiments for each EB treatment except for cytokines+BMP-4+VEGF-A₁₆₅ with n = 6). Inset represents data from similar experiments in which IL-6 or SCF was removed from the cocktail of cytokines (n = 4 with [+] or without [-] IL-6, n = 2 with [+] or without [-] SCF). In panel B, EB differentiatiation was conducted in the presence (+) or absence (-) of EPO added at the initiation of differentiation (n = 3 independent experiments). (C) Morphology under light microscopy of erythroid colonies derived from EBs differentiated with the indicated treatments. Scale bar, 100μm. (D) A representative Giemsa staining performed on 20-day pooled erythroid colonies generated from both VEGF-A₁₆₅—treated (+VEGF) and control EBs (-VEGF) (n = 2 for each EB treatment). Rare contaminating macrophages are also present. Scale bar, 100 μm.