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Epiblast stem cells are derived from the late epiblast layer of E5.5-E6.5 mice (the exact time depends on the mouse strain). Epiblast cells express pluripotent markers, such as nanog and pou5f1, but do not express zfp42. In addition, they express nodal and fgf5. In contrast to embryonic stem cells, epiblast stem cells do not have alkaline phosphatase activity. Epiblast stem cells can differentiate into the three primary germ layers both in-vitro and in-vivo.
The late epiblast layer was dissected intact from a pre-gastrula stage mouse and placed in dissociation medium in order to completely separate the epiblast layer from the extra-embryonic tissues. Then, the epiblast layers were placed in chemically-defined medium (CDM) consisting of IMDM (50% ) plus F12 NUT-MIX (50%), supplemented with insulin (7 μg/ ml), transferrin (15 μg/ ml), monothioglycerol (450 μM) and bovine serum albumin fraction V (5 mg/ ml). Medium was also supplemented with Activin A (20 ng/ml) and FGF2 (12 ng/ml). Every 4-5 days, cells were harvested and re-plated into plates pre-coated with either fetal bovine serum (FBS) or with human fibronectin (15 μg/ml).