Patent ID: 9732323
Date: 2017-08-15
CPC Classifications: A61K,A61P,C12N

Claim:
1. An in vitro differentiation process for generating mature hepatocytes from human induced pluripotent stem cells in less than 20 days, which comprises the steps of: (a) culturing human induced pluripotent stem cells (iPSCs) in MEF-conditioned medium comprising Dulbecco's modified Eagle medium (DMEM)/F12 supplemented with 20% knockout serum replacement, 10 ng/mL basic fibroblast growth factor, 1 mM L-glutamine, 100 μM nonessential amino acids, 100 μM 2-mercaptoethanol, 50 U/mL penicillin, and 50 mg/mL streptomycin to 70% confluence; (b) replacing the MEF-conditioned medium with culture medium containing 100 ng/mL activin A, 50 ng/mL wingless-type MMTV integration site family, member 3A (Wnt3a) and 10 ng/mL hepatocyte growth factor (HGF), and culturing the cells for 3-5 days; (c) replacing the culture medium with hepatic commitment medium comprising knockout [KO]/DMEM containing 20% knockout serum replacement, 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, and 1% dimethyl sulfoxide, and culturing the cells for 4-6 days; and (d) culturing cells from c) in Iscove's modified Dulbecco's medium (IMDM) containing 20 ng/mL oncostatin M, 0.5 μM dexamethasone, and 50 mg/mL insulin, transferrin, selenous acid (ITS) premix for 5-7 days to obtain differentiated hepatocytes; wherein said differentiated hepatocytes (i) exhibit gene expression profile similar to mature hepatocytes, and (ii) have function of urea production and glycogen storage.