3-Step Hepatocyte Differentiation from iPSCs

Human iPS cells (ACS-1011) were obtained from ATCC (Manassas, VA) and maintained with the stem cell culture medium SFM XF/FF (ACS-3002). The three step protocol of hepatocyte-like cells induction from iPS cells was modified on the base of previous publications [19 (link)]. First, 70% confluent iPS cells were cultured with Roswell Park Memorial Institute (RPMI)/B27 medium containing 100ng/mL activin A or 10ng/ml TGF-β1, along with 50ng/mL Wnt3a, and 10ng/mL HGF (R&D Systems) for 3 days of endodermal induction. Then, RPMI/B27 was replaced by hepatic commitment medium (DMEM containing 20% knockout serum replacement, 1mM L-glutamine, 1% nonessential amino acids, 0.1mM 2-mercaptoethanol, and 1% dimethyl sulfoxide) for another 4 days. Finally, the cells were incubated in Iscove's modified Dulbecco's medium (IMDM) supplemented with 20ng/mL oncostatin M (Invitrogen), 0.5μM dexamethasone, and 50 mg/mL ITS premix (BD Biosciences, San Jose, CA) for 5 days to induce hepatocyte maturation.