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Extra-embryonic endoderm cells were derived from E3.5 blastocysts. When grown at low density, the cell culture consists of two distinct sub-populations: a rounded, highly refractile cell type and a more stellate epithelial-like cell type. When cultured in high-density cultures, cells form a lattice-like structure.
XEN cells express extra-embryonic endoderm markers such as Hnf4 and Foxa2.
Blastocysts were plated on embryonic fibroblasts (EMFIs) feeder cells and cultured in RPMI 1640 supplemented with fetal bovine serum (20%), sodium pyruvate (1 mM), L-glutamine (2 mM ), penicillin/streptomycin (50 mg/ml each), β-mercaptoethanol (100 μM), human recombinant FGF4 (25 ng/ml ) and heparin (1 μg/ml ). Medium was changed every 3 days and after 15 days, XEN cells, observed in half the cultures, were passaged 1:1 onto new EMFIs in four-well plates. Cells are routinely cultured were routinely cultured on EMFIs in medium without FGF4 and heparin or on gelatin (0.1%), supplemented with 70% EMFI-conditioned medium . The latter is the preferred culturing method.