Expression of TGF-β superfamily signaling components in ESC-derived vascular cells. RNA samples from CCE cell–derived Flk1+ cells (FLK1+), mural cells (MC), and mixed populations of endothelial and mural cells (EC + MC) were analyzed by RT-PCR for expression of TGF-β superfamily signaling components and the housekeeping gene β-actin to normalize the amount of total cDNA in each sample. As controls, RNAs from FLK1+, MC, and EC + MC were analyzed for β-actin expression without the prior generation of cDNA, and a PCR reaction for each set of primers was run against H₂O.

Quantitative analyses of the effects of TGF-β signals on ESC-derived vascular differentiation. (a and b) Effect of TGF-β signals on proliferation of Flk1+ cells during vascular differentiation. 10⁵ Flk1+ cells derived from CCE cells were cultured in the presence (a) or absence (b) of VEGF in combination with TGF-β or SB-431542, followed by determination of cell number after 3 d. Error bars represent SD. (c) Quantitation of colony formation, endothelial and mural cell production and endothelial sheet formation. Flk1+ cells were cultured sparsely with 10% FCS in the absence or presence of VEGF in combination with TGF-β or SB-431542 for 4 d, and stained for PECAM1 and SMA. Number of colonies per well was counted to determine the effect of TGF-β signals on colony formation of Flk1+ cells. Experiments were repeated at least three times with essentially same results. Four colony types were observed; pure endothelial cells forming sheet structures (EC-sheet): pure scattered endothelial cells (EC-scattered); pure mural cells (MC); and mixed colonies consisting of endothelial and mural cells (Mix). Bars, 50 μm.

Effect of TGF-β signals on expression of claudin-5 in ESC-derived endothelial cells. (a) Levels of expression of claudin-5 and -12 in vascular cells derived from MGZ5 cells cultured in the absence or presence of VEGF in combination with TGF-β or SB-431542 analyzed by quantitative real-time RT-PCR. Error bars represent SD. (b) Claudin-5 (left, green) and PECAM1 (right, red) double immunofluorescence staining of differentiated Flk1+ cells with 10% FCS and VEGF alone (top), or in combination with TGF-β (middle) or SB-431542 (bottom). To obtain similar numbers of endothelial cells, different numbers of Flk1+ cells were plated depending on the culture conditions. Bars, 200 μm.

Effects of TGF-β superfamily members on differentiation of ESC- derived Flk1+ cells into endothelial and mural cells. (a) PECAM1 (purple) and SMA (brown) immunostaining of CCE cell–derived vascular cells. ESC-derived Flk1+ cells were treated or not with VEGF in the presence of 10% FCS. The cells were also treated with BMP7, TGF-β, activin, TGF-β + 10 μg/ml anti–TGF-β antibodies, and activin + 200 ng/ml follistatin in the presence of VEGF and 10% FCS. (b) CCE cell–derived Flk1+ cells were treated with different doses of TGF-β in the presence of VEGF and 10% FCS. Bars: (a and b) 100 μm.

Effects of various inhibitors of TGF-β superfamily members on ESC-derived vascular differentiation. (a) PECAM1 (purple) and SMA (brown) double immunostaining of differentiated Flk1+ cells derived from CCE cells with 10% FCS and VEGF in the absence or presence of follistatin, anti–TGF-β neutralizing antibodies, or both of them. (b) CCE cell–derived Flk1+ cells were treated with TGF-β and different doses of SB-431542 in the presence of 10% FCS and VEGF. (c) CCE cell–derived Flk1+ cells were treated with different doses of SB-431542 in the presence of 10% FCS and VEGF. (d) Low magnification images of PECAM1 and SMA double immunostaining of differentiated Flk1+ cells derived from CCE cells with 10% FCS in the absence or presence of VEGF in combination with TGF-β or SB-431542. (e) PECAM1 (red) and SMA (green) double immunofluorescence staining of differentiated Flk1+ cells derived from CCE cells with 10% FCS in the absence or presence of VEGF in combination with TGF-β or SB-431542. Bars: (a–c and e) 100 μm; (d) 5 mm.

TGF-β induced phosphorylation and nuclear translocation of Smad2 and Smad1/5 in ESC-derived endothelial cells. (a) Phosphorylation of R-Smads by TGF-β superfamily members in ESC- derived endothelial and mural cells. Flk1+ cells were cultured with 10% FCS and VEGF for 3 d to induce differentiation into endothelial and mural cells, and treated with TGF-β or BMP7 in the absence or presence of SB-431542 for 1 h. Mural cells differentiated from Flk1+ cells with 10% FCS were treated with TGF-β for 1 h. Cell lysates were directly subjected to immunoblotting using phospho-Smad2 (top), and phospho-Smad1/5 (bottom) antibodies. (b) Dose response of TGF-β induced Smad2 versus Smad1/5 phosphorylation. Flk1+ cells were cultured with 10% FCS and VEGF for 3 d to induce differentiation into endothelial and mural cells, and treated with different concentrations of TGF-β for 1 h. (c) Nuclear translocation of R-Smads by TGF-β and BMP in ESC-derived endothelial cells. Endothelial cells differentiated from Flk1+ cells by VEGF in serum-free culture (Yamashita et al., 2000) were untreated (left) or treated with TGF-β (middle) or BMP7 (right) for 1 h, and subjected to immunofluorescence staining using phospho-Smad2 (top) and phospho-Smad1/5 (bottom) antibodies (green) and observation by confocal microscopy. (Insets) immunofluorescence staining of the same fields for PECAM1 (red). Bars, 200 μm. (d and e) Dose response effects of SB-431542 on Smad2 phosphorylation induced by exogenous or endogenous TGF-β. Flk1+ cells were cultured with 10% FCS and VEGF for 3 d to induce differentiation into endothelial and mural cells, and treated with different concentrations of SB-431542 for 1 h in the (d) presence or (e) absence of exogenous TGF-β.

Effects of TGF-β signals on vascular differentiation of Flk1+ cells from 8.5-dpc embryos. (a) PECAM1 (purple) and SMA (brown) double immunostaining of differentiated Flk1+ cells derived from mouse embryos with 10% FCS in the absence or presence of VEGF in combination with TGF-β or SB-431542. (b) Claudin-5 (green) and PECAM1 (red) double immunofluorescence staining of differentiated embryo-derived Flk1+ cells with 10% FCS and VEGF alone, or in combination with TGF-β or SB-431542. To obtain similar numbers of endothelial cells, different numbers of Flk1+ cells were plated depending on the culture conditions. Bars: (a) 100 μm; (b) 200 μm.