Trophoblast differentiation in vitro: establishment and characterisation of a serum-free culture model for murine secondary trophoblast giant cells

Abstract

Differentiation of trophoblast giant cells is an early event during the process of murine embryo implantation. However, differentiation of secondary trophoblast giant cells in the rodent is still only partially understood, probably because of the lack of suitablein vitromodels and cell markers. In order to advance our understanding of trophoblast differentiation, suitablein vitromodels and markers are required to study their development. The objectives of this study were to establish and characterise a serum-freein vitromodel for murine secondary trophoblast cells. Secondary trophoblast giant cells growingin vitroand paraffin sections of day 8.5 postcoitum mouse embryos were processed for immunostaining to establish the expression of potential markers using antibodies to blood group antigens, E-cadherin, α7integrins and activator protein-γ, as well as placental lactogen-II. Within 3 days in serum-free culture, ectoplacental cone-derived secondary trophoblast cells underwent simultaneous induction of both morphological and functional differentiation. Secondary trophoblasts grewin vitroas a monolayer of cells with giant nuclei and expressed B and Le-b/Le-y blood group antigens, α7integrins and placental lactogen-II, as well as activator protein-γ. Transcripts for activator protein-γ and placental lactogen-II were detected in cultures by RT-PCR and for placental lactogen-II byin situhybridisation. At later time-points apoptosis increased. A fibronectin substrate significantly increased secondary trophoblast cell numbers and surface area of outgrowth. The increase in cells with giant nuclei coincided with induction of placental lactogen-II expression. A relationship was found between the nuclear area of secondary trophoblast cells and expression of placental lactogen-II.