Establishment of Embryonic Stem (ES) Cell-Like Cell Lines Derived from Bovine Blastocysts Obtained by In Vitro Culture of Oocytes Matured and Fertilized In Vitro.

Abstract

Inner cell masses (ICM) were isolated immunosurgically from 110 bovine blastocysts derived from in vitro produced day-8 embryos and subsequently cultured in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum, 10-4 M mercaptoethanol, nonessential amino acids and nucleosides on mitomycin C-treated mouse embryonic fibroblast feeder cells. Approximately 5 days after plating, growing ICMs were dissociated by trypsinization and the colonies composed of cells with ES cell-like morphology were selected for further passage at 4 to 6 days intervals. Sixty seven percent of the ICMs were clumped and 7.8% of the ICMs brought into culture produced ES cell-like cell lines. Histochemical staining revealed that all cell lines after five passages were positive for the alkaline phosphatase activity. Immunohistochemical localization of the carbohydrate antigen by 4C9 monoclonal antibody reactivity also gave a positive result similar to that seen immediately following immunodissection. Cytogenetic analysis revealed a karyotype with an euploid number of chromosomes in 48.1 to 71.0% of cells. In all cell lines, dissociated cells formed embryoid bodies 8 to 10 days after culture in the absence of feeder cells. These results suggest that the bovine ES cell-like cell lines established were pluripotent, at least in vitro.