Enhancement of early lymph vessel development in ES cells cultured in 3-dimensional collagen by inhibition of TGF-β signaling. Mouse R1 ES cells were cultured in collagen gel with VEGF-A (30 ng/mL) for the first 4 days and subsequently with VEGF-C (30 ng/mL) and VEGF-A (30 ng/mL) for 14 days to form embryoid bodies exhibiting early lymph vessel formation. The embryoid bodies were also treated with TGF-β1 (1 ng/mL) or TβR-I inhibitor (3 μM) for the last 7 days. (A,B) The embryoid bodies were stained by PECAM1 (red) and LYVE-1 (green) (A). Bars represent 50 μm. Quantification of LYVE-1–stained areas was performed in 5 low-magnification microscopic fields on 3 embryoid bodies (B). Error bars represent SD (***P < .001). (C) Immunostaining for PECAM1 (red) and Prox1 (green) confirmed the effect of modulation of TGF-β signaling on expression of Prox1 in PECAM1-positive structures.