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.
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