Cell culture
The human iPS cell line 201B7 was purchased from the RIKEN Cell Bank (Tsukuba, Japan) and cultured under feeder-free conditions in ReproFF medium (ReproCELL, Inc., Yokohama, Japan) on 10-cm dishes (Asahi Glass Co. Ltd., Tokyo, Japan) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The cells were incubated at 37°C in a humidified chamber with 5% CO2. They were then harvested with Accutase (Innovative Cell Technologies, Inc., San Diego, CA, USA), and spread onto individual 10-cm dishes at a density of 1×106 cells/dish, and passaged every 4–5 days.
Culture in conventional media with or without 3BP or 2DG
201B7 cells were harvested and spread onto 6-well plates (Asahi Glass Co. Ltd.) at a density of 1×106 cells/well and cultured in 5% of carbon dioxide at 37°C in ReproFF, Leibovitz's-15 (L15), William's E (WE) or Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DF12). Repro FF was purchased from ReproCELL Inc., all other media were purchased from Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). ReproFF, WE, and DF12 all contained glucose, whereas L15 contained. galactose. L15, WE and DF12 media were supplemented with nicotinamide (1.2 mg/ml), proline (30 ng/ml) and 10% KnockOut Serum Replacement (Life Technologies; Thermo Fisher Scientific, Inc.) medium supplement. Nicotinamide (1.2 g/l) and proline (260 mM) were added as they are necessary for primary hepatocyte proliferation (18,19). For certain experiments, 3BP (10 µM; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or 2DG (10 µM; Sigma-Aldrich; Merck KGaA) was added. Following 7 days culture, the cells were observed by light microscopy (CKX41N-31PHP; Olympus Corporation, Tokyo, Japan) without any treatment or subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
HSM
HSM was prepared from amino acid powders using the formulation of L15 medium (Life Technologies; Thermo Fisher Scientific, Inc.), but omitting arginine, tyrosine, glucose and sodium pyruvate, and with the addition of galactose (900 mg/l), ornithine (1 mM), glycerol (5 mM) and proline (260 mM; all from Wako Pure Chemical Industries, Ltd., Osaka, Japan) (11). Knockout Serum Replacement (Life Technologies; Thermo Fisher Scientific, Inc.) was used in place of fetal bovine serum to establish defined xeno-free conditions and was added at a final concentration of 10%.
RT-qPCR
Total RNA (5 µg) was isolated from 201B7 cells using Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) and subsequently used for the synthesis of first-strand cDNA with SuperScript III Reverse Transcriptase and oligo (dT) primers (Life Technologies; Thermo Fisher Scientific, Inc.), following the manufacturer's protocols. Total RNA from the human fetal liver was purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA) to serve as a positive control. qPCR was performed using Fast SYBR-Green Master Mix (Life Technologies; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and the results were analyzed using the MiniOpticon Real-Time PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, USA). qPCR was performed for 40 cycles of two steps, consisting of 5 sec denaturation (95°C) and 5 sec annealing-extension (60°C). Primer sequences are presented in Table I. The constitutively expressed housekeeping gene ribosomal protein L19 (RPL19) was used as an endogenous internal control to monitor the levels of mRNA expression (20). mRNA expression levels were analyzed automatically using the MiniOpticon system based on 2−ΔΔCq method (21). The relative expression was calculated as the expression level of a specific gene divided by that of RPL19. Experiments were repeated three times.
Plasmid construction
The SacI-HindIII fragments of the GALK1 and GALK2 promoters, contained within pLightSwitch Promoter vectors (SwitchGear Genomics, Carlsbad, CA, USA) were subcloned into the pMetLuc2-reporter vector (Promega Corporation, Madison, WI, USA) to make pMetLuc2/GALK1 and pMetLuc2/GALK2 reporter plasmids, respectively. GALK1 or GALk2 promoters (2 µg) in pLightSwitch Promoter or pMetLuc2-reporter vector, was digested with 10 U of SacI in a volume of 10 µl at 37°C for 1 h. The digested samples were mixed with 10 U of HindIII, the total volume was 50 µl. The digested fragments were fractionated with gel electrophoresis, and purified with a gel extraction kit (Qiagen GmbH, Hilden, Germany). The purified fragments were subcloned into the digested pMetLuc2-reporter with a ligation kit (Takara Bio. Inc., Otsu, Japan). The inserted fragments were confirmed by sequencing (Riken Genesis, Co. Ltd., Tokyo, Japan).
Transfection and Metridia luciferase activity assay
201B7 cells were plated on 96-well plates (Asahi Glass Co. Ltd.) coated with Matrigel, at a density of 5×105 cells/well. pMetLuc2-control, pMetLuc2/GALK1 and pMetLuc2/GALK2 were transfected with FuGENE HD Transfection Reagent (100 ng/well; Clontech Laboratories, Inc.) according to the manufacturer's protocol (22). The reporter plasmids expressed Metridia luciferase, which is secreted into the medium; the pMetLuc2-control plasmid expresses Metridia luciferase driven by the cytomegalovirus (CMV) immediate early promoter. A luciferase assay was performed following 2 days of culture in ReproFF, WE or HSM using a Ready-To-Glow Secreted Luciferase Reporter assay (Clontech Laboratories, Inc.) and a Gene Light GL-200A luminometer (Microtec Co. Ltd., Funabashi, Japan). To monitor transfection efficiency, 10 ng of the pSEAP2 control vector (Clontech Laboratories, Inc.) was added to each well of the black 96-well plates and transcriptional activity was measured using a Secreted Embryonic Alkaline Phosphatase (SEAP) Chemiluminescence kit (Clontech Laboratories, Inc.) and the Gene Light luminometer according to the manufacturer's protocols. Luciferase activity was calculated as the Metridia luciferase activity divided by the SEAP activity (relative light units). The Metridia luciferase activity was normalized against that of the CMV promoter. The Metridia luciferase activity is presented in relative light units. The experiments were repeated three times.
Statistical analysis
Relative expression levels of AFP and relative light units of Metridia luciferase were analyzed by a one-way analysis of variance using JMP version 5.0J (SAS Institute, Inc., Cary, NC, USA), followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.