Isolation of pluripotent stem cells from cultured porcine primordial germ cells

Embryonic germ (EG) cells are undifferentiated stem cells isolated from cultured primordial germ cells (PGC). To date, EG cells have been isolated only in the mouse. Murine EG cells share several characteristics with embryonic stem (ES) cells, including morphology, pluripotency, and the capacity for germ-line transmission. We report here the isolation of porcine EG cells. PGC collected from Day 24 or 25 porcine embryos were cultured on mitotically inactivated murine fibroblasts. Four EG cell lines were isolated from repeated subculture of porcine PGC. Porcine EG cells morphologically resembled murine ES cells and consistently expressed alkaline phosphatase activity. These cell lines maintained a normal diploid karyotype and survived after cryopreservation. Porcine EG cells were capable of differentiating into a wide range of cell types in culture, including endodermal, trophoblast- like, epithelial-like, fibroblast-like, and neuron-like cells. In suspension culture, porcine EG cells formed embryoid bodies. When injected into host blastocysts, the EG cells were able to differentiate and contribute to tissues of a chimeric piglet. Both in vitro and in vivo evidence demonstrates that the isolated EG cells were pluripotent. These cells are potentially useful for genetic manipulation in pigs.