Please summarise the following content:(no word
limit)
EXPERIMENTAL PROCEDURES Animals All animals including pregnant ICR mice,...
EXPERIMENTAL PROCEDURES Animals All animals including pregnant ICR mice, neonatal Wistar rats, and NOD-SCID mice (8 weeks old, male) were purchased from CLEA Japan (Tokyo). All exper- imental procedures and protocols were approved by the Animal Care and Use Committee of Keio University and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cells Mouse ESCs were obtained from the Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology. The hESC line (KhES-2 and KHES-3) was obtained from the Department of Development and Differentia- tion, Institute for Frontier Medical Sciences, Kyoto University and used in conformity with the Guidelines for Derivation and Utilization of Human Embry- onic Stem Cells of the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The hiPSC line (253G4 and 201B7) was obtained from the Center for iPSC Research and Application, Kyoto University. The skeletal myoblast cell line (C2C12), hepatocyte cell line (HepG2), and renal cell line (HEK293) were obtained from the American Type Culture Collection. Reagents The mouse monoclonal antibodies for a-actinin (immunoglobulin G (IgG,D and K167 (IgM) were purchased from Sigma-Aldrich (Sigma). The mouse monoclonal antibodies for SMA (IgGza), Bill-tubulin, Oct3/4, and Tra1-60 were purchased from Dako, Promega, BD Transduction Laboratories, and Millipore, respectively. The goat polyclonal antibodies for GATA-4 (C-20) and Nkx2.5 (N-19) were purchased from Santa Cruz Biotechnology (Santa Cruz). Alexa Fluor 488 and 546 anti-mouse IgG (IgG, IgGza, and IgM) antibody and anti-goat IgG antibody were purchased from Invitrogen. DAPI and E-cad- herin-Fc were also purchased from Invitrogen. Tetramethylrhodamine methyl ester perchlorate (TMRM), mitotracker red, 2,7'-bis-(2-carboxyethy)-5-and- 6)-carboxyfluorescein (BCECF) and Fluo-4 dye were purchased from Invitro- gen. Fibronectin, isoproterenol hydrochloride, carbamylcholine, and -CHC were purchased from Sigma. The [C]-labeled glucose and lactate were purchased from Isotec. The (14CH-labeled lactate was purchased from PerkinElmer. Maintenance of Mouse and Human PSCS We maintained mouse ESCs on gelatin-or E-cacherin-coated dishes in Glas- gow minimum essential medium ( MEM) (Sigma) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio), 0.1 mM MEM nonessential amino acid solution (Sigma), 2 mM L glutamine (Sigma), 0.1 mm B-mercaptoethanol (Sigma), and 2,000 U/ml murine leukemia inhibitory factor (Chemicon) (Nagaoka et al., 2006). We maintained hESCs and hiPSCs on MEFs in Dulbec- co's modified Eagle's medium/nutrient mixture F-12 Ham 1:1 (DMEM-F12;