Development of angiogenic embryoid bodies during one-step ES cell differentiation into type I collagen gels. A, Schematic illustration of the one-step method, performed either in 35 mm-diameter plates or in 12-well microplates, compared with the two-step initial angiogenesis model; B, Effect of the addition of angiogenic growth factors (VEGF + FGF2) on the formation of EBs exhibiting endothelial sprouts (angiogenic EBs). ES cell differentiation was performed in the absence (0) or in the presence (1) of angiogenic growth factors added at the initiation of the differentiation of CJ7 ES cells at day 0. (1+2), effect of a second angiogenic growth factor addition at day 6, in addition to day 0, on the formation of sprouting angiogenic EBs. *** p < 0.001; mean values significantly different by unpaired Student's t test. C, CD31 immunostaining of a 11 day-old angiogenic embryoid body derived from CJ7 ES cells obtained in the one-step differentiation method. The arrow points to a CD31-positive endothelial sprout (red staining) and arrowheads to CD31-negative sprouts that only exhibit blue staining of cell nuclei after Hoescht 33258 counterstaining. Scale bar = 200 μm.