OP9/ephrin-B2 promoted ECs sprouting from a single EC.

(A) Flk-1⁺PECAM-1⁻(R2) and Flk-1⁺PECAM-1⁺(R3) cells were sorted from E9.0-9.5 P-Sp regions stained with Flk-1 and PECAM-1 antibodies. (B) The sorted R2 and R3 cells were cocultured with an OP9/ephrin-B2 or OP9/vector in the presence of VEGF for 10 days, and PECAM-1 immunostaining was performed. The cordlike structures (░) from R2 and R3 cells were higher than the sheetlike structure (■) on OP9/ephrin-B2 stromal cells, but almost no difference was seen between the 2 types of structures on OP9/vector stromsl cells. (C) Expression of ephrin-B2 and EphB4 was detected by RT-PCR analysis. Total RNA was extracted from R2 and R3 cells, and the RNA extracted from E9.5 yolk sac was used as a positive control. R3 cells expressed both ephrin-B2 and EphB4, whereas R2 cells did not express either.