Recombinant mouse oncostatin m

GPSCs (129Sv/C57B (H2^b)), derived from mouse SSCs, were cultured and induced to differentiate into hepatocytes in IMDM complete media containing IMDM-Glutamax (Invitrogen), 9% FCS, 300 μmol/L mercaptoethanol, 100 U/ml penicillin, 100 μg/ml Streptomycin, 1mM sodium pyruvate, and 1x non-essential amino acids (NEAA) (Invitrogen). Feeder-free GPSCs were cultured in hanging drops (300 cells/drop) in the absence of LIF, and at Day 2, embryoid bodies (EBs) were plated on gelatin for further differentiation. The following factors were added: 20ng/ml acidic fibroblast growth factor (FGF) and 10ng/ml basic FGF from Day 6; 10ng/ml rat recombinant hepatocyte growth factor (HGF, Peprotech) from Day 10; 10ng/ml recombinant mouse oncostatin M (R&D systems), 10⁻⁷ M dexamethasone and 1x ITS solution (Sigma) from Day 16 (Fig 1A)[16 (link)]. At Day 13, EBs were trypsinised for cell sorting as described below.

At Days 18 and 21 of differentitation, one EB was plated per well on matrigel and ammonium chloride was added for 24 hours at a final concentration of 5mM in IMDM complete media supplemented with 10ng/ml recombinant mouse oncostatin M (R&D systems), 10⁻⁷ M dexamethasone and 1x ITS solution (Sigma). Urea production was determined at various time points using a colorimetric assay (Quantichrom) following the manufacturers’ instructions.