193CHARACTERIZATION OF PORCINE TROPHECTODERM-DERIVED CELLS IN THE PRESENCE OF HBMP4 IN THE ABSENCE OF A FEEDER LAYER

A porcine trophoblastic cell line could provide a powerful model for understanding trophoblast cell biology as well as placental gene expression and proteomics in vitro. In this experiment, we derived porcine trophoblastic cells from trophectoderm tissue and assessed their growth on three different extracellular matrix substrates and in three different concentrations of human recombinant bone morphogenetic protein 4 (hBMP4). Human BMP4 has been shown to induce differentiation of human embryonic stem cells into trophoblast lineages. Elongated embryos were flushed using DPBS supplemented with 1% fetal calf serum and penicillin-streptomycin (1X) from the hysterectomized uteri of superovulated and bred prepuberal gilts 15 days post-insemination. The embryonic disc was visualized with a dissecting microscope. The trophectoderm tissue was cut 2–3mm away from the embryonic disc with a scalpel and the trophectoderm tissue was manually dissected into cell aggregates. These aggregates were plated on collagen type IV, Matrigel, and human extracellular matrix (laminin, collagen type IV and heparan sulfate proteoglycan derived from human placenta) in culture medium (DMEM with 15% FCS, 0.1mM 2-mercaptoethanol, 4ngmL−1 basic FGF4 and 1X P/S) in the presence or absence of hBMP4 at 0, 10, or 20ngmL−1. Cell outgrowth was observed within 24 hours of culture. After three days of culture, various cell types (based on size and morphology) were present. Among cultures of predominant large cells were colonies of smaller cells with epithelial-type morphology that had a prominent nucleus and a high nuclear-to-cytoplasmic ratio. The epithelial-type cells grew in tight colonies with definite borders and contained cytoplasmic structures resembling lipid-containing vesicles. These colonies initially appeared on all matrices across all hBMP4 concentrations. After seven days in culture the colonies developed distinct differences across groups. Cell growth on collagen was comprised of tight colonies having definite borders among large cells. Colonies on collagen were larger and more pronounced in both the hBMP4-supplemented groups than when cultured without hBMP4. The Matrigel coated plates contained large sheets of epithelial-type cell growth instead of compact colonies. This type of growth characteristic was present in all hBMP4 treatments on Matrigel. In contrast, few cells survived and propagated on human extracellular matrix. Only small colonies having the desired morphology were among the large cells on human extracellular matrix when cultured in medium containing 10ngmL−1 hBMP4. Cells were passaged and only cells growing on Matrigel could be further cultured. These data suggest that both the cell substrate and hBMP4 affect initial trophoblast outgrowths. Further analysis including immunocytochemistry and RT-PCR is currently being performed to better characterize these cells. Epidemiology/Diseases