Fig. 5.
Analysis of ALCAM expression in endothelial precursor cells and examination of development of endothelial clusters in vitro.

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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