All experiments handling human cells and tissues were performed in line with the Tenets of the Declaration of Helsinki. Human cells in this study were performed in full compliance with the Ethical Guidelines for Clinical Studies (Ministry of Health, Labor, and Welfare, Japan). The cells were banked after approval of the Institutional Review Board at the National Institute of Biomedical Innovation (May 9, 2006). All experiments handling animals were performed in line with Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions (Ministry of Education, Culture, Sports, Science and Technology, Notice No. 71, 2006). Animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of the National Research Institute for Child Health and Development.
Viral vector construction and viral transduction
Construction of the lentiviral vector plasmids CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1, and CSII-TRE-Tight-CDK4R24C was previously described20. In brief, the EF1a promoter in CSII-EF-RfA was replaced with a tetracycline-inducible promoter, TRE-Tight, from pTRE-Tight (Clontech, Mountain View, CA) to generate CSII-TRE-Tight-RfA. Human cyclin D1, human mutant CDK4 (CDK4R24C: an INK4a-resistant form of CDK4), and TERT were inserted into the entry vector via a BP reaction (Invitrogen, Carlsbad, CA). These segments were then recombined with CSII-TRE-Tight-RfA through an LR reaction (Invitrogen) to generate CSII-TRE-Tight-cyclin D1, CSII-TRE-Tight-CDK4R24C, and CSII-TRE-Tight-TERT. The rtTA segment from pTet-Off Advanced (Clontech) was amplified by PCR, recombined with the donor vector pDONR221 via a BP reaction (Invitrogen) to generate pENTR221-Tet-Off, and then recombined with a lentiviral vector, CSII-CMV-RfA, through an LR reaction (Invitrogen) to generate CSII-CMV-Tet-Off. Recombinant lentiviruses with vesicular stomatitis virus G glycoprotein were produced as described previously21. Primary hepatocytes were inoculated with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C lentiviruses at the multiplicity of infection of 3 to 10 in the presence of 4 µg/mL of polybrene.
The cells were isolated from a liver of 4-year-old girl with biliary atresia (Hep2018) after liver transplantation. Informed consent was obtained from the parent of the patient. The hepatocytes were isolated according to the collagenase perfusion method, as described elsewhere1. The samples were minced into small pieces, washed with HEPES buffer (pH 7.7; 140 mM NaCl/2.68 mM KCl/0.2 mM Na2HPO4/10 mM Hepes), and digested with 0.5 mg/mL collagenase/DMEM (Boehringer Mannheim) diluted in the same buffer supplemented with 0.075% CaCl2 under gentle stirring at 37 °C. The cell suspension was washed twice in Hepes buffer and resuspended in an HCM BulletKit medium (cat. CC-3198, LONZA) supplemented with 10% FBS, hEGF, transferrin, hydrocortisone, BSA, ascorbic acid, insulin, Fungizone, 100 units/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, and 5 × 10–7 M hydrocortisone hemisuccinate. Cell suspensions containing 2.2 × 106 cells per vial were frozen once for future use. The frozen cells were later thawed and seeded on four 35-mm dishes (BD Falcon 6-well plate) with feeder cells of irradiated human bile duct mesenchymal cells in the ESTEM-MN medium (GlycoTechnica Ltd.) [modified F-medium (DMEM:F12(1:3), 5% FBS, 8.4 ng/ml Cholera Toxin, 30.5 μg/ul Adenine HCl, 10 ng/ml EGF, 0.4 μg/ml Hydrocortisone, 5 μg/ml human insulin, 1% Pen/Strep)] supplemented with 10 μM Y-27632 and 5% conditioned media containing Wnt3a and RSpo1. Y-27632 was supplemented in each passage. Twenty days after plating, several colonies appeared, and nine colonies with hepatocyte-like morphology was picked up by using stainless cloning cylinder with trypsin. The isolated cells were re-plated on a 35-mm dish without any coating feeder layer, and one of them (clone #9) was inoculated with recombinant lentiviruses constitutively expressing TERT and conditionally expressing Cyclin D1 and mutant CDK4 on the same day20. Cells proliferated constantly after infection, and were frozen at passage 3. These cells were maintained in the ESTEM-EP medium, and the medium was renewed every 2 or 3 days, and were passaged every 1 week (1/5 dilution) by trypsinization. The established cells were designated as HepaMN cells. HepaMN cells were passaged every 1 week (1/5 dilution) by trypsinization. HepaMN cells were maintained for 1 weeks in the ESTEM-MN medium (GlycoTechnica Ltd.) at 37℃ in 5% CO2, and the ESTEM-MN medium was renewed every 2 or 3 days.
Calculation of population doublings
Primary hepatocytes and HepaMN cells were seeded at 0.8 to 1.0 × 106 cells/well in a 100-mm dish. When the cells reached subconfluency, both primary cells and HepaMN cells were harvested and the total number of cells in each well was determined using the cell counter. Population doubling (PD) was used as the measure of the cell growth rate. PD was calculated from the formula PD 1/4 log2 (A/B), where A is the number of harvested cells and B is the number of plated cells.
Whole-cell protein extracts were used for analysis, and immunoblotting was conducted as described previously 22. Antibodies against CDK4 (clone D9G3E; Cell Signaling Technology, Danvers, MA), CyclinD1 (clone G124-326; BD Biosciences, Franklin Lakes, NJ), GAPDH (clone 6C5, Ambion), p16INK4a (clone G175-405, BD Biosciences) were used as probes, and horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulins (Jackson Immunoresearch Laboratories, West Grove, PA) were employed as secondary antibodies. The LAS3000 charge-coupled device (CCD) imaging system (Fujifilm Co. Ltd., Tokyo, Japan) was employed for detection of proteins visualized by Lumi-light Plus Western blotting substrate (Roche, Basel, Switzerland).
Histological analysis of HepaMN cells
HepaMN cells were harvested with a cell scraper and collected into tubes. The cells were analyzed with an iPGell kit (GenoStaff, Tokyo, Japan).
HepaMN cells were fixed in 4% paraformaldehyde for 10 min at 4 °C. After washing with PBS and treatment with 0.1% Triton-X100 for 10 min at room temperature, cells were pre-incubated with blocking buffer (1% BSA in PBS) for 30 min at room temperature, and then reacted with primary antibodies in blocking buffer for 12 h at 4 °C. Followed by washing with PBS, cells were incubated with secondary antibodies; anti-goat or anti-mouse IgG conjugated with Alexa 488 or 546 (1:1000) (Invitrogen) in blocking buffer for 30 min at room temperature. Then the cells were counterstained with DAPI and mounted.
Karyotypic analysis was contracted out at Nihon Gene Research Laboratories Inc. (Sendai, Japan). Metaphase spreads were prepared from cells treated with 100 ng/mL of Colcemid (Karyo Max, Gibco Co. BRL) for 6 h. The cells were fixed with methanol:glacial acetic acid (2:5) three times, and dropped onto glass slides (Nihon Gene Research Laboratories Inc.). Chromosome spreads were Giemsa banded and photographed. A minimum of 10 metaphase spreads were analyzed for each sample, and karyotyped using a chromosome imaging analyzer system (Applied Spectral Imaging, Carlsbad, CA).
RNA was extracted from cells using the ISOGEN (NIPPON GENE). An aliquot of total RNA was reverse transcribed using an oligo (dT) primer (SuperScript III First-Strand Synthesis System, Invitrogen). For the thermal cycle reactions, the cDNA template was amplified (QuantStudio 12K Flex Real-Time PCR System) with gene-specific primer sets (Supplemental Table 1) using the Platinum Quantitative PCR SuperMix-UDG with ROX (11743–100, Invitrogen) under the following reaction conditions: 40 cycles of PCR (95 °C for 15 s and 60 °C for 1 min) after an initial denaturation (50 °C for 2 min and 95 °C for 2 min). Fluorescence was monitored during every PCR cycle at the annealing step. The authenticity and size of the PCR products were confirmed using a melting curve analysis (using software provided by Applied Biosystems) and gel analysis. mRNA levels were normalized using ubiquitin or GAPDH as a housekeeping gene.
Gene chip analysis
High quality total RNA was isolated from each cell using Trizol following the manufacturer’s instructions (Invitrogen, USA). Genomic DNA was eliminated by treatment with DNAse I for 20 min at RT using DNAse IH (Invitrogen, USA). RNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware USA). Purity and integrity of total RNA was determined by 260/280 nm ratio and checked by electrophoresis in Bioanalyzer RNA6000 Nano. About 100 ng of total RNA was used to produce Cyanine 3-CTP labeled cRNA using the Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies) according to the manufacturer’s instructions. Following ‘One-Color Microarray Based Gene Expression Analysis’ protocol version 6.0 (Agilent Technologies), 2 μg of labeled cRNA was hybridized with a human gene expression microarray 60 K (Agilent Technologies, Santa Clara CA, USA). The microarray workflow quality control was implemented using the Agilent Spike-In Kit which consisted of a set of 10 positive control transcripts optimized to anneal to complementary probes on the microarray with minimal self-hybridization or cross-hybridization. The concentrated Agilent One-Color RNA Spike-In mix stock was diluted in the buffer provided by the kit and mixed with the RNA samples prior to the amplification and labeling process to achieve the relative amounts recommended by the manufacturer. For hybridization, Agilent gene expression microarray 60 K slide (Design ID: 72,363. SurePrint G3 Human Gene Expression 8 × 60 K Microarray Kit, Agilent Technologies) were used. Slides were scanned in an Agilent C Scanner according to the manufacturer’s protocol. Signal data were collected with dedicated Agilent Feature Extraction Software (v 11.5.1). Agilent Processed Signals were processed using GeneSpring software (Agilent Technologies).
Principal component analysis (PCA)
To analyze the expression data of genes in an unsupervised manner with a gene chip array, we used principal component analysis23,24. PCA, a multivariate analysis technique which finds major patterns in data variability, was performed to categorized HepaMN cells into each stage of ESCs undergoing hepatic differentiation. The differentiation protocol was previously reported in detail25. For ESC-derived hepatocytes, Sees2 was maintained in the XF32 medium [85% Knockout DMEM, 15% Knockout Serum Replacement XF CTS (XF-KSR), 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, 50 µg/mL l-ascorbic acid 2-phosphate, 10 ng/mL heregulin-1β (recombinant human NRG-β 1/HRG-β 1 EGF domain), 200 ng/mL recombinant human IGF-1, and 20 ng/mL human bFGF. To generate embryoid bodies (EBs), ESCs (1 × 104/well) were dissociated into single cells with Accutase after exposure to the rock inhibitor (Y-27632), and cultivated in the 96-well plates in the EB medium [76% Knockout DMEM, 20% Knockout Serum Replacement, 2 mM GlutaMAX-I, 0.1 mM NEAA, Pen-Strep, and 50 µg/mL l-ascorbic acid 2-phosphate for 10 days. The EBs were transferred to the 24-well plates, and cultivated in the XF32 medium for 14 to 35 days.
In vivo analysis
The operation protocols were approved by the Laboratory Animal Care and the Use Committee of the National Research Institute for Child and Health Development, Tokyo. HepaMN cells were harvested with a cell scraper, collected into tubes, centrifuged, and resuspended in PBS (200 μl). The same volume (200 μl) of Matrigel was added to the cell suspension. The cells (> 1 × 107) were subcutaneously inoculated into severe combined immunodeficiency (SCID) mice (CREA, Tokyo, Japan). After 9 days, the resulting tumors were dissected and fixed with PBS containing 4% paraformaldehyde. Paraffin-embedded tissue was sliced and stained with hematoxylin and eosin (HE). HepaMN cells were also implanted into spleen in immunodeficient SCID mice. HepaMN cells were harvested with a cell scraper, collected into tubes, centrifuged, and resuspended in PBS (50 μl). The cells (> 1 × 106) were intrasplenically implanted. After 7 days, the spleens were dissected and fixed with PBS containing 4% paraformaldehyde for further histological analysis.
Subcutaneous nodules on mouse backs were fixed in 20% formalin and embedded in paraffin. Cut paraffin sections were deparaffinized, dehydrated, and treated with 2% proteinase K (Dako) in Tris–HCl buffer solution (pH 7.5) for 5 min at room temperature, or heated in ChemMate Target Retried Solution (Dako) for 5–20 min in a high-pressure steam sterilizer for epitope unmasking. After washing with distilled water, samples were placed in 1% hydrogen peroxide/methanol for 15 min to block endogenous peroxidase. The sections were then incubated at room temperature for 60 min in primary antibodies diluted with antibody diluent (Dako). The following primary antibodies against various human differentiation antigens were used: vimentin (V9, M0725, Dako, Glostrup, Denmark), albumin (Dako), AE1/AE3 (712811, NICHIREI), and Ki67 (ABCAM, ab15580). Then, they were washed three times with 0.01 M Tris buffered saline (TBS) solution (pH 7.4) and incubated with goat anti-mouse or anti-rabbit immunoglobulin labeled with dextran molecules and horseradish peroxidase (EnVision, Dako) at room temperature for 30 min. After three times washes with TBS, they were incubated in 3,3′-diaminobenzidine in substrate-chromogen solution (Dako) for 5–10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.
CYP3A4 induction test
HepaMN and HepaRG cells were examined in the 24-well plates in modified F-medium [DMEM:F12(1:3), 5% FBS, 8.4 ng/ml cholera toxin, 30.5 μg/ul adenine HCl, 10 ng/ml EGF, 0.4 μg/ml hydrocortisone, 5 μg/ml human insulin, 1% pen/strep], and was treated with 20 μM rifampicin or 125 μM omeprazole for 24 h at a cell density of 80%. Controls were treated with DMSO (final concentration 0.1%).
CYP3A4 activity and chromatographic conditions
The reaction mixtures [0.05 M potassium phosphate buffer (pH 7.4), 2 mM NADPH (Trevigen Inc., MD), 20 μM 7-benzyloxy-4-trifluoromethylcoumarin (BFC, Sigma-Aldrich, MD) and cell homogenate (20–100 μg protein) in a final volume of 200 μl] were incubated at 37 °C for 1 h26. Reactions were terminated at 95 °C for 2 min, followed by centrifugation at 10,000 × g for 5 min. For the inhibition assay by ketoconazole (KTZ, Cayman Chemical Co., Michigan, USA) as inhibitor of human CYP3A isoform, KTZ in 50% methanol solution, yielding final concentrations 10 μM in the incubation mixture, was added to the incubation tubes27. A Coomassie Plus (Bradford) Protein Assay kit (Thermo Fisher Scientific, MA, USA) was used to determine protein concentrations with bovine serum albumin as the standard.
Quantitative analysis of 7-Hydroxy-4-trifluoromethylcoumarin (HFC) produced by CYP3A4 was performed by reverse-phase high performance liquid chromatography system (RP-HPLC) equipped with a Cosmosil 5C18-AR-II column (4.6 I.D. × 150 mm, Nacalai Tesque, Kyoto, Japan). RP-HPLC analysis was carried out in isocratic conditions using a reciprocating dual pump KP-22 (FLOM Inc., Tokyo, Japan) and a Rheodyne manual injector with a loop size of 100 μl. Running conditions include: injection volume, 50 μl; mobile phase, methanol: 0.02 M phosphate buffer (pH7.4) (3:4 v/v); flow rate, 0.2 ml/min; and fluorescence detection at an excitation wavelength of 410 nm and an emission wavelength of 510 nm using a Multi λ Fluorescence Detector 2475 (Waters Co., Milford, MA). Calibration curves known amounts of HFC (Sigma-Aldrich, MD) were added to the mixture of buffer–methanol (1:1). The linear concentration range was 1 to 100 nmol/ml for HFC.
Statistical analysis was performed using the unpaired two-tailed Student’s t test.