Hepatocyte-like Cell Differentiation Protocol

Differentiation of hPSCs into hepatocyte-like cells was induced by a modified four-step induction method [4 (link)]. Briefly, suspended cells were seeded onto polystyrene plates coated with Matrigel, human recombinant vitronectin, R-Fc, or NC-Fc. Before confluency, the cells were treated with RPMI-1640 medium containing 100 ng/ml Activin A and B27 supplement (without insulin; Life Technologies) for 5 days in a normoxic atmosphere (20% O₂). The cells were then specified into the hepatic lineage by treatment with RPMI-1640/B27 (with insulin) medium containing 10 ng/ml bFGF and 20 ng/ml BMP4 for 5 days in a hypoxic atmosphere (4% O₂). After specification, the cells were cultured in RPMI-1640/B27 medium with 20 ng/ml hepatocyte growth factor for 5 days (4% O₂) and then in HCM (without epidermal growth factor; Lonza, Basel, Switzerland) supplemented with 20 ng/ml oncostatin M for 5 days (20% O₂).