Fig. 1. Effects of TGFβ family members on mouse ESC-derived endothelial cells (MESECs). (A-E) MESECs were obtained by CD34-sorting of vascular (endothelial and mural) cells derived from mouse ESCs, and cultured in the presence of 10% fetal calf serum (FCS) and VEGF (A), followed by immunofluorescence staining for PECAM1 (red) and SMA (green). MESECs were also treated with BMP4 (B), TGFβ2 (C), activin (D) and TβR-I inhibitor (E). Scale bars: 100 µm. (F) Quantitative analysis of the effects of TGFβ signals on colony formation from single MESECs. MESECs were cultured at low density with 10% FCS and VEGF in the absence (–) or presence of TGFβ2 (Tβ) or TβR-I inhibitor (inhib) for 4 days, and then stained for PECAM1 and SMA. The number of colonies per well was counted to assess the effect of TGFβ signals on colony formation of MESECs. Two colony types were observed: those consisting of pure endothelial cells (E) and those also containing mural cells (M). Experiments were repeated at least three times with essentially the same results.