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Epiblast stem cells were derived from the epiblast of post-implantation mouse embryos. Cells formed teratomas in-vivo which contained cells of all the three germ layers. Cells have a normal karyotype (40,XY).
E5.5 mouse embryos were dissected out from their decidua layer, into HEPES-buffered medium. In order to isolate the epiblast layer, an incision was made at the boundary between the extra-embryonic ectoderm and the epiblast layers. The embryonic fragment consisting of the epiblast and the overlying visceral endoderm layer was digested in a solution of trypsin and pancreatin in PBS and subsequently returned to the initial HEPES-buffered dissection medium. The isolated epiblast fragments were then cultured on a feeder cell layer in DMEM/F12, supplemented with knockout serum replacement (20%), FGF2 (5 ng/ml), 2-mercaptoethanol (0.1 mM ), L-glutamine (2 mM), and non-essential amino acids (X1).