Figure 6.

Migration assay using HUVEC to confirm that VEGF secreted from HESC is biologically active. HESC were treated with RA (1 μm) + TPA (50 nm) or solvent control for 6 h, then washed and refed without added compounds, and cultured overnight. This procedure generated high VEGF-containing supernatant without the continued presence of RA and TPA. The migration assay was performed as described in Materials and Methods. Briefly, 2 × 10⁶ HUVEC were plated on Matrigel-coated transwell filters and conditioned media from vehicle- (panel A) or RA + TPA-pretreated (panel B) HESC were added to the lower chambers. After 24 h, HUVEC migrating toward the conditioned media were stained with Giemsa. The marked increase in HUVEC migration in chambers containing supernatant from the RA + TPA-treated cells reflects the substantial up-regulation of bioactive VEGF secretion.