Hepatic Differentiation Protocol from Cells

Hepatic differentiation was performed following the protocol described in [20 (link)]. Briefly, when cells reach 70% confluency, the medium was changed for RPMI1640 media containing B27 Supplements Minus Insulin (Invitrogen), 100 ng/mL Activin A (R&D Systems), 20 ng/mL fibroblast growth factor 2 (FGF2) (R&D Systems), and 10 ng/mL bone morphogenetic protein 4 (BMP4) (PeproTech) to induce endoderm. After 8 days of culture, dishes were moved to hypoxia (4% O₂) in RPMI/B27 Supplement (Invitrogen) medium with 20 ng/mL BMP4 and 10 ng/mL FGF2 for 5 days. Next, the medium was changed to RPMI/B27 supplemented with 20 ng/mL hepatocyte growth factor (HGF, PeproTech) for an additional 5 days in hypoxia. The final stage of differentiation was in HCM hepatocyte culture medium (Lonza, but omitting the EGF) supplemented with 20 ng/mL Oncostatin M (R&D Systems) for 5 days in normoxia (21% O₂). During that time, the medium was freshly prepared and changed daily.