IN-VITRO CULTURE OF EMBRYONIC DISC CELLS FROM PORCINE BLASTOCYSTS

The aim of the present study was to define the conditions of preparati on and in vitro culture of embryonic discs allowing proliferation of E S-like cells. G5-6 porcine blastocysts (GO = day of Al) were cultured in toto; in G1O-11 blastocysts, trophectoderm and primitive endoderm w ere microsurgically removed from embryonic discs (ED) which were cultu red either on plastic or on a feeder layer. Feeder cells were foetal G 30 porcine fibroblasts which had been previously irradiated. Culture m edium was DMEM supplemented with 0.1 mM beta-mercaptoethanol, 5% foeta l calf serum, 5% Ultroser G and 10(3) IU LIF; cultures were performed at 38 degrees C. Colonies were reseeded weekly. Few embryonic discs fr om G5-6 and no elongating blastocysts gave rise to ES-like cells. At l east 50% G10-11 ED attached and developed multilayered colonies (100 c ells) of small ovoid ES-like cells. Colonies from 4 sows were maintain ed in culture for at least 8 wk. Addition of PDGF, insulin or both, in duced 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 ret inol induced differentiation of ES-like cells. These cells introduced in nude mice induced teratoma.