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For h-HepOrg hepatic differentiation, h-HepOrgs were expanded in EM2 medium above, split, seeded and cultured for 25 days under EM1 culture medium, after which the medium was changed to DM medium composed of AdDMEM/F12 supplemented with 1% HEPES, 1% GlutaMAX, 1% penicillin/streptomycin, 1 B27 without retinoic acid, 1.25 mM N -acetylcysteine, 50 ng ml 1 hEGF, 15% RSPO1 conditioned medium, 50 ng ml 1 HGF, 2 M A83-01, 3 M CHIR99021, 10 M Y-27632, 0.5 nM Wnt surrogate Fc fusion protein, 100 ng ml 1 FGF19 (R&D Systems) and 1.6 M dexamethasone (Sigma)