title: Establishment and characterization of first trimester human trophoblast cells with extended lifespan.
abstract: We established trophoblast cell cultures with extended lifespans by introducing into first trimester human trophoblasts the gene encoding simian virus 40 large T antigen. The transfected trophoblasts were characterized according to their expression of various morphological and functional markers. Both parental (HTR-8) and transfected (HTR-8/SVneo) lines were morphologically similar and positive for cytokeratin, confirming their epithelial (trophoblastic) identity. Whereas the parental cells senesced after 12-14 passages, the transfectants have been in culture for over 32 passages. Human chorionic gonadotrophin was detected only in the HTR-8/SVneo cells and not in the parental cells. Both lines required at least 5% serum in order to sustain growth in vitro and responded to transforming growth factor-beta (TGF-beta) with reduced [3H]-thymidine incorporation in a dose-dependent manner. Treatment with TGF-beta also resulted in decreased secretion of plasminogen activators (PAs) and reduced PA activity by both lines. Both cell lines secreted mostly 72-kDa type IV collagenase as determined by substrate gel zymography, but the level of secretion of this enzyme was not significantly affected by TGF-beta in either line. Even though both lines exhibited similar in vitro invasive abilities, only the invasiveness of the parental cells was reduced by TGF-beta. Neither parental or transfected cells were capable of growth in soft agar and no sign of tumor formation was evident more than 5 months after subcutaneous inoculation of the transfected cells into nude mice. These results indicate that apart from their ability to sustain prolonged growth in culture, the transfected HTR-8/SVneo cells share a number of phenotypic properties with the parental trophoblast cells. For this reason, these transfected trophoblasts may prove to be an important tool for the study of placental function and/or tumor progression.
authors.affiliation: Division of Cancer Research, Sunnybrook Health Science Centre, Toronto, Ontario, Canada.
authors.forename: C H
authors.initials: CH
authors.lastname: Graham
authors.forename: T S
authors.initials: TS
authors.lastname: Hawley
authors.forename: R G
authors.initials: RG
authors.lastname: Hawley
authors.forename: J R
authors.initials: JR
authors.lastname: MacDougall
authors.forename: R S
authors.initials: RS
authors.lastname: Kerbel
authors.forename: N
authors.initials: N
authors.lastname: Khoo
authors.forename: P K
authors.initials: PK
authors.lastname: Lala
mesh_terms: D000818:Animals; D002455:Cell Division; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods; D004261:DNA Replication; D010450:Endopeptidases / Q000032:analysis / Q000378:metabolism; D005260:Female; D018093:Gelatinases; D006801:Humans; D007633:Keratins / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008819:Mice, Nude; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D013539:Simian virus 40 / Q000235:genetics; D013936:Thymidine / Q000378:metabolism; D014162:Transfection / Q000379:methods; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014183:Transplantation, Heterologous; D014327:Trophoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D006063:Chorionic Gonadotropin; D003470:Culture Media; D016212:Transforming Growth Factor beta; D007633:Keratins; D010450:Endopeptidases; D018093:Gelatinases; D013936:Thymidine