Before the initiation of hepatic differentiation, human iPSCs were dissociated into single cells by using TrypLE Select Enzyme and plated onto BD Matrigel Basement Membrane Matrix Growth Factor Reduced (BD Biosciences). The hepatic differentiation protocol was based on a previous report with some modifications (55 (link)). Briefly, in definitive endoderm differentiation, human iPSCs were cultured with the RPMI 1640 medium (Sigma-Aldrich) containing activin A (100 ng/ml; R&D Systems), 1% GlutaMAX (Thermo Fisher Scientific), and 1× B27 Supplement Minus Vitamin A (Thermo Fisher Scientific) for 4 days. For the induction of hepatoblast-like cells, the definitive endoderm cells were cultured with RPMI 1640 medium containing bone morphogenetic protein 4 (20 ng/ml) (R&D Systems) and fibroblast growth factor 4 (20 ng/ml) (R&D Systems), 1% GlutaMAX, and 1× B27 Supplement Minus Vitamin A for 5 days. To perform hepatic differentiation, the hepatoblast-like cells were cultured for 5 days in RPMI 1640 medium (Sigma-Aldrich) containing hepatocyte growth factor (HGF; 20 ng/ml), 1% GlutaMAX, and 1× B27 Supplement Minus Vitamin A. Last, the cells were cultured for 11 days in hepatocyte culture medium (Lonza) without EGF but with oncostatin M (20 ng/ml).