Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture

Abstract

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.