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Term decidual tissue consists of terminally differentiated decidual cells, bone marrow-derived cells, and fibroblast cells. Since undifferentiated decidual cells are fibroblast-like cells of the endometrial stroma, the possibility exists that the fibroblast cells in term decidua are undifferentiated decidual cells. To test this hypothesis, a purified population of fibroblast cells was isolated from term decidua and treated under conditions that are known to induce differentiation of endometrial stromal cells. By flow cytometry and immunocytochemistry, the fibroblast cells from term decidua were shown to be free of cells expressing bone marrow-derived cell-surface antigens and the epithelial cell marker cytokeratin. In addition, they tested positive for the cytoskeletal protein vimentin, thus establishing that they were mesenchymal cells. As with endometrial stromal cells, continuous treatment of the decidual fibroblast cells with the progesterone analog medroxyprogesterone acetate and estradiol in combination with either dibutyryl-cAMP or prostaglandin E2 induced cell aggregation and the expression of prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). When cells were plated at an initial cell density of 0.25 x 10(6) cells/well in a 24-well culture dish with medium changes every three days, PRL was first detected on Days 4-6, and the peak of averaged 24 h-PRL release (30 ng/well) occurred on Days 26-28. The mRNA for decidual fibroblast PRL followed a temporal pattern corresponding to that of the released hormone. The size of the PRL mRNA was 1.15 kb, corresponding to the alternately spliced PRL mRNA reported for decidualized endometrial stromal cells and other extrapituitary sources of PRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Under appropriate conditions, protease-dispersed cytotrophoblast cells from human term placenta fuse and differentiate into syncytiotrophoblast cells that express hCG. Although human placental lactogen (hPL) is also expressed by the syncytiotrophoblast in vivo, little hPL is expressed by trophoblast cells differentiated in vitro using standard tissue culture medium or keratinocyte growth medium supplemented with fetal bovine serum. In this report, we demonstrate that cytotrophoblast cells fuse and differentiate to a phenotype that expresses large amounts of both hCG and hPL when cultured in medium containing maternal serum. When protease-dispersed cytotrophoblast cells were plated in RPMI-1640 supplemented with 10% second trimester (16-22 week) maternal serum (STMS medium), extensive cell fusion was observed by day 3 in culture. Cell fusion appeared to be essentially complete by day 6, coinciding with the peak of hCG expression and the initiation of hPL expression. The peak of hCG expression occurred between days 4-7 of an experiment; the range for maximal release of hCG was 0.1-3.7 micrograms/24 h.well when cells were plated at a density of 10(6)/well. In contrast, the peak of hPL expression occurred between days 7-11 of an experiment, and the range of maximal hPL release was 0.9-3.5 micrograms/24 h.well. Northern blot analysis indicated that the level of hPL messenger RNA (mRNA) paralleled the release of hormone. However, whereas the hCG beta-subunit mRNA paralleled the release of hCG, the hCG alpha-subunit mRNA did not, suggesting that the two genes are independently regulated. Cells cultured in nonpregnant (male or female) serum-supplemented medium also differentiated to the syncytiotrophoblast phenotype following a temporal pattern almost identical to that observed for cells cultured in STMS. However, the quantity of hormone released was at least 2-fold greater with STMS. Compared to RPMI-1640 or keratinocyte growth medium supplemented with 10% fetal bovine serum, STMS induced equal or greater expression of hCG and substantially greater expression of hPL at the level of both mRNA and protein. The extent of cell fusion, the level of hormone expression, and the regulated patterns of hormone expression suggest that cytotrophoblast cells cultured in maternal serum progress to an advanced stage of trophoblast differentiation.