Embryonic stem cells expressing both platelet endothelial cell adhesion molecule-1 and stage-specific embryonic antigen-1 differentiate predominantly into epiblast cells in a chimeric embryo

We examined the expression of cell-surface markers on sub-populations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripoten-cy. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule I (PE-CAM-1) and stage-specific embryonic antigen (SSEA)-l in ES cells. Almost all SSEA-1 (+) cells expressed a high level of PECAM-1, and reversible repopulation was observed between PECAM1(+)SSEA-1(+) and PECAM-1(+)SSEA-1(-) cells. The ES cells carrying the IacZ gene were sorted into three subpopulations: PECAM-1 (-)SSEA-1(-), PECAM-1 (+)SSEA-1 (-), and PECAM-1 (+)SSEA-1 (+). Quantitative reverse transcription-polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1 (-) cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1(+) SSEA-1(+/-) cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM-1 - cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0-7.0 days post coitum, many PECAM-1 (+)SSEA1 (+) cells were found in the epiblast, but few beta-gal(+) cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.