Embryonal carcinoma cells differentiate into parietal endoderm via an intermediate stage corresponding to primitive...

F9 embryonal carcinoma (EC) cells were used as a model system to study endoderm formation during mammalian embryogenesis. F9 cells treated with retinoic acid (RA) or RA plus dibutyryl cyclic AMP (cAMP) were examined for the expression of stage-specific embryonic antigen-3 (SSEA-3), a cell surface marker of primitive and visceral endoderm. SSEA-3 was not detected by indirect immunofluorescence on the surface of undifferentiated stem cells; however, a subset of SSEA-3-positive cells appeared with time in culture, amounting to 20% of cells 10 days after plating. When cultured in the presence of RA, the percentage of SSEA-3-positive cells increased to 70% of cells 10 days after plating. In contrast, treatment of cells with RA plus cAMP yielded differentiated cells that were SSEA-3-negative. These SSEA-3-negative cells exhibited ultrastructural features of parietal yolk sac endoderm. In contrast, SSEA-3-positive cells appearing in cultures treated with RA alone exhibited ultrastructural features of primitive endoderm on day 3, switching to ultrastructural features of parietal endoderm on day 10. Cells with hybrid features, resembling both visceral and parietal yolk sac, were also seen. We suggest that differentiation of F9 EC cells into parietal yolk sac-like cells can occur along two distinct pathways: 1) direct under the combined influence of RA and cAMP; and 2) indirect, under the influence of RA alone, in which cells first differentiate into primitive endoderm. Parietal yolk sac-like cells induced through the latter pathway continue to express SSEA-3, a cell surface marker of primitive endoderm that is not normally found on parietal endodermal cells in vivo.