Fig. 5.
Fig. 5. 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.

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.

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