Collagen 1 coated 6 well plates

Hepatocyte differentiation was performed as previously described15 (link); briefly, the induction of definitive endoderm (DE) from hESCs was initiated by RPMI medium (Invitrogen, Carlsbad, CA) with 100 ng/ml Activin A (R & D Systems Inc., Minneapolis, MN) without serum for 2 days, and then the medium was changed to RPMI medium with 100 ng/ml Activin A, 0.5 mM sodium butyrate and B27 supplement (Invitrogen) for up to 6 days. DE cells were then split and re-seeded on collagen I-coated 6-well plates (BD Biosciences, San Diego, CA) in hepatocyte differentiation medium (HDM) which contains IMDM media (Invitrogen) supplemented with 20% FBS (Invitrogen), 2 mM L-glutamine (Invitrogen), 0.3 mM 1-thioglycerol (Sigma-Aldrich, St. Louis, MO), 0.5% DMSO (Sigma), 100 nM dexamethasone (Sigma), 0.126 U/ml human insulin (Hospira, Inc), FGF-4 (20 ng/ml), HGF (20 ng/ml), BMP2 (10 ng/ml), and BMP4 (10 ng/ml) (R & D Systems Inc.) for 2 weeks. Then the cells were further differentiated and maturated in hepatocyte culture medium (HCM) which contains hepatocyte basal medium (Lonza, Walkersville, MD) supplemented with SingleQuots kit (Lonza), 0.5% DMSO, 100 nM dexamethasone, 20 ng/ml FGF4, 20 ng/ml HGF, and 50 ng/ml oncostatin M (R & D systems Inc.) until use.

Hepatocyte differentiation started from the induction of definitive endoderm (DE) from hESCs using previously described protocols [21] (link). Briefly, hESCs was induced to DE in RPMI medium (Invitrogen) with 100 ng/ml activin A (R & D Systems Inc.) without serum for 2 days and then the medium was changed to RPMI medium with 100 ng/ml activin A, 0.5 mM sodium butyrate and 1×B27 supplement (Invitrogen) for 6 days. DE cells were then split and re-seeded on collagen I-coated 6-well plates (BD) in IMDM media (Invitrogen) supplemented with 20% FBS (Invitrogen), FGF-4 (20 ng/ml), HGF (20 ng/ml), BMP2 (10 ng/ml), BMP4 (10 ng/ml) (R and D), 0.3 mM 1-thioglycerol (Sigma), 0.5% DMSO (Sigma), 100 nM dexamethasone (Sigma) and 0.126 U/ml human insulin (Hospira, Inc) for 2 weeks. Then the cells were further differentiated and maturated in hepatocyte culture medium supplemented with SingleQuots (Lonza), 0.5% DMSO, 100 nM dexamethasone, 20 ng/ml FGF4, 20 ng/ml HGF, and 50 ng/ml oncostatin M (R & D systems) until use.