Fig. 1. VEGF stimulates primitive erythropoiesis. (A) ES cells were differentiated into EBs in either the presence or absence of VEGF, which was added on day 3 (d3). The hematopoietic precursor content of EBs was assessed by plating dissociated cells in the presence of hematopoietic growth factors (See Materials and methods). LIF, leukemia inhibitory factor; Ery^P, primitive erythroid colonies. (B-E) Morphology of day-7 EBs. Vegf^–/– ES cells were differentiated for 7 days in either the absence (B,C) or presence (D,E) of VEGF (5 ng ml^–1). Note the larger size and the intensity of hemoglobinization in VEGF-treated EBs. (F) EB size was estimated by integrating individual surface area using Northern Eclipse software. Histograms illustrate the distribution of individual EB area. The range covered by columns is equivalent and determined arbitrarily. Data were analyzed by Student's t-test: –VEGF, n=102; +VEGF, n=44; P<0.001. (G) VEGF stimulates a dose-dependent increase in the number of primitive erythroid progenitors. Vegf^–/– ES cells were differentiated in the presence of increasing concentrations of VEGF and assayed for primitive erythroid precursors (Materials and methods). Results are the mean±s.d. of duplicates and are representative of five independent experiments. (H) Morphology of day 4 Ery^P. (I) Morphology of colony cells revealed by Wright-Giesma stain. Scale bars: 100 µm in B-E,H; 10 µm in I.