B27 supplement minus vitamin a

Human iPS cells were dissociated into single cells with TrypLE Select Enzyme and seeded on Matrigel (growth factor reduced; Corning, NY, USA)-coated plates. Differentiation of human iPS cells into hepatocytes was performed based on our previous methods with some modifications.²⁰ (link) Briefly, in the first step, human iPS cells were cultured in RPMI1640 medium (Sigma-Aldrich) with 1x GlutaMAX (Thermo Fisher Scientific), 0.5x B27 Supplement Minus Vitamin A (Thermo Fisher Scientific), and 100 ng/mL Activin A (R&D Systems, MN, USA) for 4 days to induce definitive endoderm cells. In the second step, definitive endoderm cells were cultured in RPMI1640 medium with 1x GlutaMAX, 0.5x B27 Supplement Minus Vitamin A, 20 ng/mL bone morphogenetic protein (BMP) 4 (R&D Systems) and 20 ng/mL fibroblast growth factor (FGF) 4 (R&D Systems) for 5 days to induce hepatoblast-like cells. In the third step, hepatoblast-like cells were cultured in RPMI1640 medium with 1x GlutaMAX, 0.5x B27 Supplement Minus Vitamin A, and 20 ng/mL hepatocyte growth factor (HGF; R&D Systems) for 5 days to HLCs. In the fourth step, the cells were cultured in hepatocyte culture medium (HCM; Lonza, Basel, Switzerland) containing 20 ng/mL oncostatin M (OsM; R&D Systems) without epidermal growth factor for 11 days for maturation.

Before the initiation of hepatic differentiation, human iPS cells were dissociated into single cells by using TrypLE Select Enzyme and plated onto Matrigel‐coated dishes. The cells were then cultured in StemFit AK02N medium for 24 hours. The differentiation protocol for the induction of definitive endoderm cells, hepatoblast‐like cells, and hepatocyte‐like cells was based on our previous reports with some modifications.11 Briefly, in the definitive endoderm differentiation, human iPS cells were cultured for 4 days in Roswell Park Memorial Institute 1640 (RPMI1640) medium (Sigma‐Aldrich), which contained 100 ng/mL Activin A (R&D Systems), 2× GlutaMAX, and 0.5× B27 Supplement Minus Vitamin A (Thermo Fisher Scientific). For the induction of hepatoblast‐like cells, the definitive endoderm cells were cultured for 5 days in RPMI1640 medium containing 20 ng/mL BMP4 (R&D Systems), 20 ng/mL fibroblast growth factor 4 (FGF4; R&D Systems), 2× GlutaMAX, and 0.5× B27 Supplement Minus Vitamin A. To perform hepatic differentiation, the hepatoblast‐like cells were cultured for 5 days in RPMI1640 medium containing 20 ng/mL hepatocyte growth factor, 2× GlutaMAX, and 0.5× B27 Supplement Minus Vitamin A. Finally, the cells were cultured for 11 days in hepatocyte culture medium (Lonza) without EGF but with 20 ng/mL oncostatin M and 3× GlutaMAX.