FIG. 1.

Endothelial growth media-2 (EGM-2) stimulates vascular endothelial differentiation of fetal lung mesenchymal cells. (A) Immunoblot for the SV40 Large T antigen shows degradation over time after culture at 37°C. β-actin included as loading control. (B) E15 cells were cultured in DMEM containing 10% fetal bovine serum (left panels) or endothelial growth media (EGM-2; right panels). Phase contrast micrographs of representative fields show that cells cultured in EGM-2 acquire a more flattened, cobblestone morphology. (C) At day 7, cells cultured in EGM-2 (right panel) had increased Flk-1 expression as measured by FACS. Control cells cultured in DMEM (left panel) did not express Flk-1 above background (labeled with nonimmune IgG). (D) Immunostaining of mesenchymal cells shows increased cell surface VE-cadherin in cells cultured for 7 days in EGM-2 and loss of α-SMA stress fibers. Staining for α-SMA in EGM-2 cultured cells was seen in a more cytoplasmic pattern. (E) Cells cultured for 7 days in DMEM or EGM-2 were then plated on Matrigel. Bright field images taken 18 h later show increased tube formation in EGM-2 cultured cells. DMEM, Dulbecco's modified Eagle's medium; α-SMA, α-smooth muscle actin.