OP9/ephrin-B2 supported SMC recruitment, whereas OP9/EphB4 and EphB4-Fc inhibited it.

(A) The vascular networks in the P-Sp explant cultures were visualized by PECAM-1 staining (blue), and the anti–α-SMA antibody was used to detect the SMCs on the P-Sp culture system after coculture for 14 days. The α-SMA⁺ (red) cells were located more abundantly in the vascular bed on OP9/ephrin-B2 stromal cells than in OP9/vector stromal cells (arrowheads indicate the α-SMA⁺ cells), and almost no α-SMA⁺ (red) cells were detected in the OP9/EphB4 stromal cells. The bar indicates 100 μm. (B) Schematic presentation of SMC recruitment under various conditions. Figures correspond to the upper panels in panel A. (C) SMA⁺ cells were derived from P-Sp explants. P-Sp explants from embryos of green mice expressing GFP ubiquitously (i-iii) and wild-type embryos (iv-vi) were cocultured with OP9 stromal cells, and the culture plates were immunostained with Cy3-conjugated anti–α-SMA antibodies. Green indicates cells from GFP P-Sp explants; red indicates α-SMA⁺ cells; and yellow indicates GFP and α-SMA⁺⁺ cells. Panels Ci and Civ are fluorescein isothiocyanate (FITC) specific, Cii and Cv are rhodamine (RHOD)–specific wavelengths, and the merged configuration is shown in panels Ciii and Cvi. The bar indicates 25 μm.