Lymphatic and blood vessel endothelial cell development from Tie2^-/- ES cells. (A) Flk1⁺ cells (2000) were cultured on OP9 cells. At day 7 of culture, the number of vascular endothelial colonies developed from Tie2^+/- (□) and Tie2^-/- (▪) ES cells was calculated. (B) Vascular endothelial colonies were stained with PECAM-1 mAb and analyzed at low (× 40) and high (× 100) magnification. (C) The number of lymphatic endothelial colonies developed from Tie2^+/- (□) and Tie2^-/- (▪) ES cells was calculated. (D) Lymphatic endothelial colonies were stained with LYVE-1 mAb and analyzed at low (× 40) and high (× 100) magnification. Scale bars in panels B and D, 20 μm. In the presence of Tie2-Fc (30 μg/mL), the number of vascular and lymphatic endothelial colonies was decreased (panels E and F, respectively). (G) Hematopoietic cluster formation was not affected by exogenous soluble Tie2-Fc (30 μg/mL). Exogenous soluble CD4-Fc (30 μg/mL) served as the control. Results in panels A, C, and E-G are expressed as the mean ± SD.