Purinergic receptor activation by agonist stimulation of Rb efflux from human placental cytotrophoblast cells in culture

BeWo cells derived from a choriocarcinoma have been extensively used as a model of placental trophoblast. There are several clones of the BeWo cell line. Authentication and characterisation of these cloned cell lines is important to ensure their identity and purity prior to use in research. In order to be classified as cytotrophoblasts, cells need to fulfil certain criteria including the expression of cytokeratin-7 and secretion of human chorionic gonadotrophin (hCG). BeWo cells were obtained from ATCC (hereafter referred to as the ATCC clone) and from A.L. Schwartz, St Louis Children’s Hospital, St Louis, Missouri, USA (hereafter referred to as the Schwartz clone). Cells in culture were maintained in Ham’s F12 and DMEM, supplemented with 10 % fetal calf serum and 2 % penicillin/ streptomycin. Aliquots of both clones were sent to the European Collection of Cell Cultures for DNA profiling which indicated 75 and 80 % homology between the original cell line and the ATCC and Schwartz clones, respectively. Cells were seeded onto glass coverslips and immunocytochemistry was used to visualise cytokeratin-7 and vimentin. Collagen-coated aluminium oxide filters were seeded with 2 w 10 cells per filter and after 4 days cells were exposed to forskolin for 48 h. Media samples were collected and assayed for hCG. Polycarbonate filters were seeded and cells grown for 10 days. The transepithelial electrical resistance (TEER) was measured in order to determine if cells formed a polarised monolayer. Cells from both clones stained with the anti-cytokeratin-7 antibody. The ATCC clone but not the Schwartz clone showed reactivity with the anti-vimentin antibody. hCG was detectable in media samples from ATCC BeWo at (mean ± S.E.M.) 341 ± 200 international units per litre (U l) and Schwartz BeWo at 437 ± 184 U l (n = 6 for each clone) and increased following exposure to forskolin to 6363 ± 1289 U l (n = 3) in ATCC BeWo and 9746 ± 47 U l (n = 3) in Schwartz BeWo. The Schwartz clone formed a confluent monolayer on the polycarbonate filters and generated a TEER of 42.6 ± 16.6 V cm (n = 18). The ATCC clone did not generate a TEER. These results demonstrate that the ATCC and Schwartz clones differ in characteristics such as monolayer formation and sensitivity to forskolin but they express trophoblast specific markers and undergo biochemical differentiation after exposure to forskolin. Both clones are derived from the parent BeWo line and can be used as trophoblast models.