PDGF-BB promotes vascular development and sprouting angiogenesis by PDGFR-β. (A) Embryoid bodies derived from R1 or Pdgfr-β^–/– stem cells were cultured for 8 days in the absence (basal) or presence of 30 ng/mL PDGF-BB. Endothelial cells were visualized by staining for CD31 (red; bars represent 300 μm). Immunoblotting for PDGFR-β was performed on embryoid bodies cultured in the absence (–) or presence (+) of PDGF-BB. (B) Embryoid bodies cultured for 8 days in the presence PDGF-BB were exposed to neutralizing PDGFR-β antibody CD140b or control nonimmune serum (Ctrl) between day 6 and day 8 of differentiation. Immunostaining for VE-cadherin showed the formation of a capillary plexus in cultures treated with the control antibody but not in cultures treated with the neutralizing antibody (arrows). Bars represent 300 μm. (C) Embryoid bodies of wild-type and Pdgfr-β^–/– origin were placed in 3-D collagen gels at day 4 and cultured in the presence of VEGF-A165 (30 ng/mL) until day 10. Embryoid bodies were whole-mount stained for expression of CD31 (red), α–SMA (green), and Hoechst 33342 (blue). Bar represents 50 μm.