Vascular endothelial cells (ECs) and most hematopoietic cells express platelet endothelial cell adhesion molecule-1 (PECAM-1), which is the cell surface protein also expressed in mouse embryonic stem (ES) cells. To better understand how PECAM-1+ ES cells differentiate into PECAM-1+ hematopoietic cells/ECs, 3 cell surface markers, PECAM-1, stage-specific embryonic antigen-1 (SSEA-1), and Flk-1, were utilized to dissect the developmental process during ES cell differentiation in vitro. Undifferentiated ES cells expressed PECAM-1, with a majority of them coexpressing SSEA-1. During ES cell differentiation, expression of PECAM-1 decreased to give rise to PECAM-1−/SSEA-1+ cells, which represented epiblast stem cells. Subsequently, Flk-1–expressing cells developed from PECAM-1−/SSEA-1+ cells, becoming SSEA-1−/Flk-1+ through the downregulation of SSEA-1 expression. Following this, a second wave of PECAM-1 expression, which represented the mature hematopoietic cells/ECs, developed from Flk-1+ cells. Also, a small portion of PECAM-1+/SSEA-1+ cells, which represented the residual undifferentiated ES cells, were consistently observed in long-term differentiated embryoid bodies. This work revealed a sequential change in PECAM-1, SSEA-1, and Flk-1 expression during ES cell differentiation; therefore, they could be valuable cell surface markers for isolating cells at distinct developmental stages in ES cell differentiation.
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