Characterization of pure human first-trimester cytotrophoblast cells in long-term culture: Growth pattern, markers, and hormone production

Abstract

Pure long-term cytotrophoblast cultures were established from human first-trimester placentas by growing chorionic villus explants without enzymatic digestion. Cytoplasmic human chorionic gonadotropin was detectable in all (100%) cells in culture when labeled with a polyclonal anti-human chorionic gonadotropin antibody and in 71% to 83% of cells labeled with a monoclonal anti-alpha-human chorionic gonadotropin antibody. Most of the cells expressed cytokeratin and surface Trop-1 and Trop-2 antigens (89% to 95%), but none expressed cytoplasmic vimentin or surface 63D3 antigens. Study of the ultrastructure of the cells demonstrated epithelial morphologic features. The average doubling time of the trophoblast was 48 to 96 hours. Some of the lines have been continuously propagated for 8 months. They produced variable amounts of human chorionic gonadotropin (50 to 710 mIU/ml per 10(5) cells per 24 hours). The basal level of progesterone secreted by trophoblast (444.4 +/- 32.4 pg/ml per 10(5) cells per 24 hours) doubled in the presence of pure human chorionic gonadotropin (100 ng/ml). They produced small amounts of 17 beta-estradiol (less than 20 pg/ml per 10(5) cells per 24 hours); human chorionic gonadotropin had no effect on the estradiol production. Trophoblast-derived human chorionic gonadotropin acted as a growth factor because trophoblast proliferation (measured by uptake of thymidine labeled with tritium) was reduced by 60% in the presence of an anti-human chorionic gonadotropin antibody. Availability of pure, functionally competent human cytotrophoblast in long-term cultures is relevant for further studies in reproduction biology.