001. ISOLATION AND CHARACTERISATION OF PORCINE ES CELLS

Abstract

Embryonic stem (ES) cells have the capacity for self renewal, can remain undifferentiated in long term culture and can contribute to all the cells in the body including the germ cells. ES cells have been isolated in mice and have also been described for humans. However despite considerable effort for more than two decades ES cells which can contribute to the germline are yet to be isolated for the pig or any domestic species for that matter. We have developed a new method for isolating porcine ES cells which uses whole embryos cultured in alpha MEM with 10% serum replacement plus additives under 5% O₂. Unlike methods employed previously this method results in homogenous outgrowths whose cells resemble ES cells and which express Oct 4 and Nanog and SSEA-1 [1]. These cells can be passaged and cryopreserved repeatedly resulting in the establishment of cell lines at similar efficiencies to that reported previously for 129Sv mice [2]. These cells can form embryoid bodies and can be differentiated to various cell types representative of all three germ layers [3]. Following their injection into blastocysts these cells localise /become incorporated in the inner cell mass and can be used to produce chimaeras when these embryos are transferred to recipient animals [2]. To date we have produced chimaeric pigs from one male ES cell line [2]. These are currently being mated to demonstrate germline transmission. Future studies will examine the applicability of our method to other species commencing with mice and cattle before extending these to humans.