Analysis of ALCAM expression in endothelial precursor cells and examination of development of endothelial clusters in vitro.

(A) E10.5-derived AGM cells (upper column) or yolk sac cells (lower column) were fractionated into Flk-1⁺CD31⁺cells. Flk-1⁺CD31⁺ cells were subdivided at the expression of ALCAM, and ALCAM⁻ or ALCAM⁺cells were sorted individually for further experiments. Among CD34⁺c-Kit⁺ cells in E10.5-derived AGM, 7.6% were Flk-1⁺; however, they were ALCAM⁻ (data not shown). (B,C) A total of 750 AGM- or yolk sac–derived Flk-1⁺CD31⁺ALCAM⁺ or Flk-1⁺CD31⁺ALCAM⁻ cells were cultured with OP9 cell in the presence of VEGF for 9 or 10 days. Culture was fixed and stained with anti-CD31 antibody. Two types of CD31⁺ clusters (sheetlike and cordlike structure) were observed under a microscope (B). Macroscopic observation clearly demonstrated that distribution of endothelial clusters developed on OP9 cell (magnification, ×6.3) (C). Note that the larger CD31⁺endothelial clusters were observed in both sheetlike and cordlike clusters when ALCAM⁺ endothelial precursor cells were cocultured with OP9 cell. Scale bar, 100 μm. (D) The number of CD31⁺ sheetlike (left) or cordlike (right) clusters derived from AGM and yolk sac was scored. (E) AGM-derived endothelial precursor cells (Flk-1⁺CD31⁺) were cultured with OP9 cell in the presence of ALCAM-Fc or control CD4-Fc, and the number of CD31⁺ endothelial clusters was scored (left, sheetlike cluster; right, cordlike cluster). *P < .05.