VEGF-A₁₆₅ increases the proportions of early progenitors/precursors during EB differentiation. EBs were differentiated in the presence of cytokines+BMP-4 (-VEGF) or cytokines+BMP-4+VEGF-A₁₆₅ (+VEGF) for the indicated days and analyzed by flow cytometry for coexpression of CD34 and KDR (A-C) as well as of c-kit and Ep-CAM (D-E) and CD34 and glycophorin A (F-G). For panels A, D, and F, dot plot quadrants were based on their isotype controls shown as insets. (A) Represents a flow cytometric analysis performed at day 15 of EB differentiation. Mean fluorescence intensities (MFIs) for KDR expression are indicated. (B) Histograms represent the mean percentage of the CD34⁺/KDR⁺ subset in both types of EBs at the indicated days of differentiation, with the corresponding SD (vertical bars) (n = 7 experiments for day 15 and n = 3 for both days 0 and 7). At day 15, the proportion of cells coexpressing CD45 within both CD34⁺/KDR⁺ populations are represented by gray bars. (C) Plot showing the straight line fit for the predominance of the CD34⁺/KDR⁺ subset at day 15 of VEGF-A₁₆₅—treated EBs versus the increase in the number of erythroid colonies derived from these EBs. (D, F) Representative flow cytometric analyses of Ep-CAM+/c-kit+ and CD34+/glycophorinA+ coexpressions respectively, performed on control (-VEGF) and VEGF-A₁₆₅—treated (+VEGF) EBs differentiated for 7 days. (E, G) Kinetics analyses of the mean percentages of the above subsets at the indicated days of EB differentiation. SD are represented by vertical bars (n = 3 to 5 independent experiments for each subset and day). (H) Represents the mean percentages of the Ep-CAM+/c-kit+ subset within EBs differentiated for 15 days with the indicated treatments in the presence (+) or absence (-) of EPO. SD are represented by vertical bars (n = 3 independent experiments).