Expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 of embryonic stem cells during vasculogenesis and angiogenesis

Objective: To study the expression of alternatively spliced isoforms of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) of embryonic stem cells (ES cells) during vasculogenesis and angiogenesis. Methods: Mouse ES cells of the line J1 were cultured. Another ES cells were cultured in differentiation medium to induce the formation of embryonic bodies (EBs). Then the ES cells with PECAM-1 and EBs were inoculated with methylcellulose into Petri dish, containing cell growth factor, VEGF, bFGF, EPO, and IL-6 and the ES cells cultured for 11 days were inoculated in the Petri dish with collagen for 72 hours so as to induce sprouting angiogenesis. Immunofluorescence analysis, RT-PCR, and flow cytometry were used to detect the expression of PECAM-1, Oct-4, and stage-specific embryonic antigen (SSEA)-1 in the undifferentiated ES cells, EBs, and EB sprouting. In order to delineate the alternatively spliced cytoplasmic domain isoforms of PECAM-1 specific primers were designed to span the exon-exon junctions in the regions of alternative splicing. In order to amplify the cytoplasmic domains of all possible PECAM-1 isoforms a sense primer spanning the border of exons 9 and 10 within the cytoplasmic domain and an antisense primer spanning the border of exon 16 and 3′-untranslated region were used. Then the PCR products of the cytoplasmic domain underwent subsequent sequencing to analyze the expression of the 8 known alternatively splice isoforms of PECAM-1. Results: The ES cells expressed high level PECAM-1 that was mainly located at the cell-cell junctions. The SSEA-1 and Oct-4 levels rapidly decreased along with the differentiation of the ES cells. All 8 known alternatively splice isoforms of PECAM-1 were expressed in the ES cells and the EB sprouting, the expression of Δ14% 15 and Δ12&14&15 being the highest. The expression level of Δ12&14&15 increased markedly and the expression of Δ15 decreased along with the differentiation of ES cells. Conclusion: PECAM-1 is a constitutive feature of undifferentiated ES cells. Its changes in splice form mark the differentiation and may participate in vasculogenesis and angiogenesis.