Regulation of human extravillus trophoblast (EVT) cell differentiation and proliferation in vitro-role of epidermal growth factor (EGF)

Abstract

Summary First trimester chorionic villus explants, maintained on growth factor reduced Matrigel in serum-free media, were used to assess the effect of exogenous EGF on EVT cell proliferation and hCG production in vitro . It has been previously demonstrated that villus explants maintained on Matrigel give rise to EVT cell outgrowth from the tips of anchoring villi. Proliferating cell nuclear antigen (PCNA) antibodies were used to identify dividing cells in both non-cultured, cultured control, and EGF treated placental villus tissue. In non-cultured tissue from 5–6 weeks of gestation, PCNA immunostained most of the nuclei of the villus and cell column cytotrophoblast cells. The percentage of PCNA positive cytotrophoblast cells was reduced to about 70–80% in late gestation (8–10 weeks) first trimester tissue. PCNA failed to immunostain nuclei in syncytiotrophoblast. In both control and EGF treated cultures, PCNA immunostaining after 4 days in vitro was observed in most nuclei of villus and cell column cytotrophoblast and in 90% of cytokeratin positive cells that have migrated into the matrix outside the villus. These cytokeratin positive, mononucleated cells have been previously identified as EVT cells differentiated in vitro . The number of EVT cells differentiated in vitro has lower in late than early gestation villus tissue cultures. After 6 days in culture, the number of PCNA positive EVT cells was reduced to about 60%. Most immunostaining was observed in EVT cells close to the tips of anchoring villi. The complete disappearance of PCNA immunostaining was observed in cultures maintained in vitro for 10 and more days (results not shown). EGF supplemented culture media (1, 10, and 100 ng/ml) did not stimulate EVT proliferation and did not prevent the gradual decrease of PCNA positive cells. In contrast to the lack of effect on EVT proliferation, EGF produced strictly gestational age dependent effects on hCG production. Exogenous EGF stimulated in a dose dependent manner (1–10 ng/ml) hCG production by explants of 8–10 weeks of gestation and was without effect on hCG production by explants of 5–6 weeks of gestation. As exogenous EGF did not affect the percentage of PCNA positive cells and the number of EVT cells in villus explants 5–6 and 8–10 weeks of gestation, it was concluded that this growth factor is not mitogenic for EVT cells in vitro . The possibility that EGF alone or with some other growth factors may act through an autocrine mechanism is not, however, excluded.