In vitro culture of embryonic disc cells from porcine blastocysts

The aim of the present study was to define the conditions of preparation and in vitro culture of embryonic discs allowing proliferation of ES-like cells. G5-6 porcine blastocysts (G0 = day of AI) were cultured in toto; in G10-11 blastocysts, trophectoderm and primitive endoderm were microsurgically removed from embryonic discs (ED) which were cultured either on plastic or on a feeder layer. Feeder cells were foetal G30 porcine fibroblasts which had been previously irradiated. Culture medium was DMEM supplemented with 0.1 mM beta-mercaptoethanol, 5% foetal calf serum, 5% Ultroser G and 10(3) IU LIF; cultures were performed at 38 degrees C. Colonies were reseeded weekly. Few embryonic discs from G5-6 and no elongating blastocysts gave rise to ES-like cells. At least 50% G10-11 ED attached and developed multilayered colonies (100 cells) of small ovoid ES-like cells. Colonies from 4 sows were maintained in culture for at least 8 wk. Addition of PDGF, insulin or both, induced a transitory stimulation of growth in G6 or G10-11 ED; TGF beta did not modify growth of G6 ICM. Uterine G10-11 flushing medium or retinol induced differentiation of ES-like cells. These cells introduced in nude mice induced teratoma.