VE-cadherin–mediated cell-cell adhesion of Flk-1⁺ cell clusters on OP9 feeder layer. (A) Sorted Flk-1⁺ cells were cultured on OP9 feeder layer in the presence of blocking MoAb against VE-cadherin (VECD1). After 3 days of culture, immunostaining with MoAb for Flk-1 was performed as described in Materials and Methods. Cell-cell contact was disrupted by this treatment, whereas the proliferation of Flk-1⁺ cells was not affected. (B, C, and D) Sorted Flk-1⁺ cells were cultured on OP9 feeder layer. After 3 days, the cultures were left untreated (B) or added either with 50 ng/mL VEGF (C) or 50 ng/mL VEGF and VECD1 (D) and were further incubated for another 24 hours. Each Flk-1⁺ cell preserved cell-cell contact in culture with VEGF (C), whereas cell-cell contact of Flk-1⁺ cells was completely disrupted in the presence of VEGF and VECD1 (D). Scale bar = 100 μm.