24 Before

differentiation, the cells were cultured on Matrigel-coated tissue culture dishes using MEF-conditioned medium.25 The in vitro differentiation protocol was similar to our previously reported study and that of Hay et al.9 In brief, when human iPSCs had attained a confluence of 70%, the MEF-conditioned medium was replaced with Roswell Park Memorial Institute/B27 with 100 ng/mL activin A (PeproTech, London, UK), 50 ng/mL Wnt3a, and 10 ng/mL HGF (R&D Systems) for 3 days of endodermal induction. During the next step, the culture medium was replaced with hepatic commitment medium (knockout selleck inhibitor [KO]/DMEM containing 20% knockout serum replacement, 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM 2-mercaptoethanol, and 1% dimethyl sulfoxide). Finally, during the maturation step, the cells were culturing in Iscove’s

modified Dulbecco’s medium (IMDM) supplemented with 20 ng/mL oncostatin M (Invitrogen), 0.5 μM dexamethasone, and 50 mg/mL ITS premix (BD Biosciences, San Jose, CA). Details of the materials and methods used are described in the Supporting Information. Five- to 8-week-old NOD-SCID mice were purchased from National Laboratory Animal Center (Taipei, Taiwan). All the experimental Sirolimus datasheet procedures involving the use of animals were approved by the Animal Care Committee of the Taipei Veterans General Hospital. The lethality of CCl4 on NOD-SCID mice was tested by gavage. Hepatocyte-like cell transplants were performed at 24 hours after administration of CCl4 by intrasplenic injection, as previously reported.26 RNA was isolated from human iPSCs and human iPSC-derived hepatocyte-like cells, using

the RNeasy kit (Qiagen). Complementary DNA synthesis, fragmentation, hybridization, washing, staining, and scanning were Ribose-5-phosphate isomerase performed at the National Research Progress for Genomic Medicine Microarray and Gene Expression Analysis Core Facility, National Yang-Ming University VYM Genome Research Center, Taiwan. To provide a visual impression of how the various sample groups are related, principal component analysis (PCA) was performed using the Partek Genomics Suite program (Partek Inc., St. Louis, MO). Array data of control iPSCs and differentiated hepatocyte-like cells were downloaded from the GEO database (accession number GSE14897).17 Human iPSC and ES cell colonies were plated onto a MEF feeder layer for several months with weekly passaging. Before hepatogenic differentiation, cells were passaged using Matrigel-coated feeder-free culture conditions.