Generation of liver organoids

All cell types were incubated at 37°C in a humidified 5% CO₂ environment. iPSCs (clone code TkDA3) were kindly provided by K. Eto and H. Nakauchi (Tokyo University). Undifferentiated human iPSCs were maintained in mTeSR medium (Stemcell Technologies) on Matrigel (Corning)-coated feeder-free plates. HUVECs and MSCs were purchased from Lonza and maintained in Endothelial Growth Medium (EGM) or MSC Growth Medium (Lonza). Protocols for endoderm differentiation, hepatic specification, and liver organoid formation were as described previously (Takebe et al., 2014). Briefly, for definitive endoderm differentiation, iPSCs were dissociated by Accutase (Stemcell Technologies) and plated onto a Matrigel-coated dish. Medium was replaced with RPMI1640 containing 1% B27 without insulin (Life Technologies), 1 mM sodium butyrate (for the first 3 days), recombinant Wnt3a (R&D Systems; 50 ng/ml) and activin (R&D Systems; 100 ng/ml) for 5 to 6 days. For hepatic specification, definitive endoderm iPSCs (DE-iPSCs) were further treated with KnockOut DMEM (KO-DMEM) containing 20% knockout serum replacement, 1 mM L-glutamine, 1% non-essential amino acids, 0.1 mM 2-mercaptoethanol (all from Invitrogen) and 1% DMSO (Sigma) for 3 days. Hepatic-specified endoderm iPSCs (HE-iPSCs) were then dissociated with TrypLE (Gibco) and mixed with dissociated MSCs and HUVECs at a ratio of 10:2:7. The cell mixture was plated on Matrigel bed (50% dilution of neat Matrigel, solidified at 37°C for 15 min) with medium for the liver organoid self-organization culture (LO medium). The LO medium consisted of 50% Hepatocyte Culture Medium (HCM, Lonza) and 50% EGM (Lonza). HCM was supplemented with HCM BulletKit (Lonza): transferrin, hydrocortisone, BSA fatty acid free, ascorbic acid, insulin, GA-1000, omitting human epidermal growth factor. EGM was supplemented with EGM BulletKit (Lonza): bovine brain extract, hEGF, hydrocortisone, fetal bovine serum (FBS), GA-1000 and ascorbic acid. After mixing HCM and EGM, 10 ng/ml recombinant hepatocyte growth factor (Sigma), 20 ng/ml recombinant oncostatin M (R&D Systems), 100 nM dexamethasone (Sigma), and 2.5% FBS (CELLect Gold, MP Biomedicals) were added to complete LO medium.