Endocrinology, Vol 135, 321-329, Copyright © 1994 by Endocrine Society

ARTICLES

Human cytotrophoblast cells cultured in maternal serum progress to a differentiated syncytial phenotype expressing both human chorionic gonadotropin and human placental lactogen

RG Richards, SM Hartman and S Handwerger
Division of Pediatric Endocrinology, Children's Hospital, Cincinnati, Ohio.

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.

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