Differentiation of hPSCs into Hepatocytes

hPSCs were differentiated as discussed previously (Si-Tayeb et al. 2010 (link); Mallanna and Duncan 2013 ; Yanagihara et al. 2016 (link)). Briefly, hPSCs were harvested using Accutase and plated at 600,000 cells per well in 24-well plates precoated with 300 µl/well Geltrex (Thermo Fisher Scientific, Waltham, MA). Approximately 24 h after seeding the cells with mTeSR1 (Stemcell Technologies), when the cells were 85–95% confluent, differentiation was initiated by culture for 5 d with 50 ng/ml Activin A (R&D Systems) in RPMI/B27 (without insulin) supplement (Invitrogen) under ambient oxygen/5% CO₂. In addition, we included 10 ng/ml BMP4 (R&D Systems) and 20 ng/ml FGF2 (R&D Systems) for the first 2 d. Then, the cells were cultured for 5 d with 20 ng/ml BMP4 (R&D Systems)/10 ng/ml FGF-2 (R&D Systems) in RPMI/B27 (containing insulin) under 4% O₂/5% CO₂, then for 5 d with 20 ng/ml HGF (R&D Systems) in RPMI/B27 (containing insulin) under 4% O₂/5% CO₂, and finally for 5 d with 20 ng/ml Oncostatin-M (R&D Systems) in Hepatocyte Culture Media (Lonza) supplemented with SingleQuots (without EGF) in ambient oxygen/5% CO₂.