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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;

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| Animals :- Experimental procedures and protocols proved by the Animal care and use comitte. Examples - pregnent ICR nice , neonatal Wistar rats, and NOD-SCID mice purchased from CLEA Japan - CELLS :- Mouse ESCs obtained from RIKEN center and KHES-2 and khes-3 obtained from Frontier Medical Sciences The hipsc line (253G4 and 20137) obtained from Center of ipse Research and Application. The Skeletal myoblast cell line (C2C, 12) hepatocyte cell line (HepG2), and renal cell line (HEK293) Ostained from American Culture Collection. REAGENTS:- (is Mouse monoclonal antibodies for a-actinin and Ki 67 (IgM) purchased from sigma - Aldrich. (1) Mouse monoclonal antibodies for SMA (196r2al. Plll-tubulin , Oct 314 and Tra 1-60 purchased from Dako, Millipore ,BD Pransduction Laborator (iii) The goat polyclonal antibodies - GATA 4 [(-20) Teacher's Signature

and NKX 2.5 (1-19) purchased from Cruz Biotechnology Alexa Fluor 488 and 546 anti-mouse IgG (IgG, Ig Gro, and IgH) antibody and anti goat IgG antibody purchased from Invotrogen les Fibronectin, isoproterenal hydrochloride, Carbamylcholine, and & - CHe purchased from Sigma. (W) The [130] - faseled glucose and lactate purchased from Isotee. (vii) The [1967 - labeled lactate purchased from Perkin Elmer. MAINTENANCE OF MOUSE AND HUMAN PSCS W Mouse ESCs con gelatin or E-Cad herin-coated dishes with 10% fetal bovine serum 044 0.1mM MEM solution non essential amino acid (1) 2mML glutamine i 0.1mm .p- mercaptoe thandl

inhibitory 2,000 ulme murine leukemia factor : (vi) 207 Knockout serum. CD 0.1 mM MEM non- essential amino acid solution x 2mm glutamine (x) Olmm B- mercaptoethanol. (xi) 4 ng/ml basic fibroblast growth factor DIFFERENTIATION OF HUMAN PSC - DERIVED CARDIOMYOCYTES Undifferentiated colonies of hESCs and hipses with a MEM containing 50 ug/ml. ascorbic acid, with 5% FBS and 0.1 mm primet mercaptoeth anal in bacterial petri dish. Beating cells are observed 14-20 days later. PURIFICATION OF HESC- and hipsc - DERIVED CARDIO MYOCYTES W Glucose-free DMEM supplemented with 4mm lactate medium produced using IM lactate stock solution prepared from déluting som Teacher's Signature..........

lactate with sterile IM Na-HEPES (1 a differentiation day 20-30 were washed and exposed to selection medium W Media days (1) Split Test should be changed every 2-3 using 40 tem filters pampled cell into 2 experiments: cultivation and FACS analysis (V) FACS analysis performed antibodies and in test using a MEM supplemented using a-actinin Scultivation with 5% FACSFAI av Purified Cardiomyocytes collected by rapid flushing ✓ INMUNOFLUORESCENCE i Fixed cell with 4% paraformaldehyde ein PBS for comin. (1) Cells permeabilized with 0.1% Triton x-100 at room temperature for comin. at 4°c overnight.

LACTATE FLUXOME ANALYSIS BY CAPILLARY ELECTROPHORESIS AND MASS SPECTROMETRY Modified glucose - free DNEM Cinvitrogen, supplemented with 4 mM913 7 labeled lactate . AC710N - POZENT ZAL RECORDINGS USING MICROELECTRODES W Standard glass microelectrodes had a De resistance of 25-35 mega ut filled with pipette. O Signals are monitored recorded as electronic files and analyzed offline with PCLAMP 10 software ATP MEASUREMENT cells plated onto gelatin - coated as well I white; clear -bottom Culture blate. ATP levels measured cesing an ATP assay kit. After shaking thin and încubaiting for 20 min at 23°c, measure luminescence in a luninometer. 6 Teacher's Signature