OP9/EphB4 stromal cells inhibited vascular network formation from E9.5 P-Sp explants.

(A) OP9 cells were cotransfected with expression plasmid DNA pRK5-EphB4 (OP9/EphB4) and pCAGneo or expression vector pCAGneo alone (OP9/vector), and individual G418-resistant clones were selected (OP9/EphB4 or OP9/vector). Protein expression was observed by immunoblotting using anti-EphB4 PolyAb as portrayed in Figure 1A. (B) P-Sp explants were cocultured with OP9/EphB4 or OP9/vector stromal cells. After 14 days, the cultures were immunostained by anti–PECAM-1 MoAb. The formation of vascular networks (vn) was inhibited on the OP9/EphB4 stromal cells compared with the OP9/vector stromal cells. Vascular network formation was also inhibited by the addition of EphB4-Fc in both the OP9/vector and OP9/ephrin-B2 stromal cells. The bar indicates 500 μm. (C) P-Sp explants were cocultured with OP9/ephrin-B2, OP9/EphB4, or OP9/vector stromal cells in the presence of chimeric protein ephrin-B2–Fc for 10 days. The vascular EC sprouting and remodeling from P-Sp explants was promoted by the addition of ephrin-B2–Fc, especially in OP9/ephrin-B2 stromal cells. The bar indicates 500 μm.