Immortalization of human hepatocytes from biliary atresia with CDK4R24C, cyclin D1, and TERT for cytochrome P450 induction testing

Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.


Results
Generation of HepaMN cells. Cells were isolated from a 4-year-old patient with biliary atresia (Fig. 1A-D). These cells had a hepatocyte-like morphology after primary culture, and were immortalized by the introduction of CDK4 R24C , cyclin D1, and TERT. Immortalized hepatocytes, designated as HepaMN cells, expressed CDK4 R24C , cyclin D1, and TERT (Fig. 1E,F). HepaMN cells appeared as a homogeneous cell population with an epithelial phenotype showing no regular structural organization. Even after reaching confluence, the cells retained the appearance of hepatocyte-like cells. Morphological characteristics of HepaMN cells did not significantly change even at later passages.
HepaMN cells reproducibly proliferated more rapidly than the primary cells as determined by population doubling values (Fig. 1G). HepaMN cells continued to expand, and neither cessation of cell proliferation, such as senescence nor apoptosis/cell death, was detected during culture through 21 passages (141 days), implying that the hepatocytes were immortalized by the introduction of CDK4 R24C , cyclin D1, and TERT genes (Fig. 1H). We performed immunoblot analysis of CDK4 and cyclin D1 to confirm the expression of the transgenes (Fig. 1I). We found that CDK4 and cyclin D1 were highly expressed, and suppressed by doxycycline treatment. To determine TERT, we performed a TRAP assay in a series of hepatocytes infected with the TERT gene (Fig. 1J). The results clearly show that the TERT infection resulted in the induction of enzyme activity. We also performed karyotypic analysis of HepaMN cells. HepaMN cells had a normal karyotype (7 out of 20 metaphases) and chromosome 2 trisomy (9 out of 20 metaphases) at passage 16 ( Fig. 1K-M).

Characterization of HepaMN cells in vitro.
Immunocytochemistry revealed that HepaMN cells were positive for albumin and epithelial keratins ( Fig. 2A-D). Interestingly, HepaMN cells also positively stained for the mesenchymal marker vimentin, and cells positive for both keratins (AE1/AE3) and vimentin were observed (Fig. 2E). Vimentin, a marker for a mesenchymal phenotype, is induced in cells undergoing epithelial-to-mesenchymal transition under certain conditions, such as development and oncogenesis, and indeed hepatocytes without introduction of the genes for immortalization were negative for vimentin (Supplemental Fig. 1). HepaMN cells became positive for vimentin after the immortalization process. The other immortalized hepatocytes analyzed (immortalized cells from Hep2040, Hep2023D, Hep2020, and Hep2013) were all positive for vimentin. The paraffin-embedded HepaMN cells were again positive for vimentin, and contained glycogen in the cytoplasm by PAS stain (Fig. 2F-J). Electron microscopy studies of HepaMN hepatocytes showed numerous glycogen granules in the cytoplasm (Fig. 2K,L). HepaMN hepatocytes exhibited desmosomes between adjacent cells, with structures resembling bile canaliculi, delineated by typical junctional complexes and presenting several microvilli (Fig. 2M,N). HepaMN cells contained numerous subcellular organelles, such as mitochondria, peroxisomes, lysosomes and endoplasmic reticulum (Fig. 2O,P).

Hepatocyte-associated gene expression in HepaMN cells. HepaMN cells were monitored by ana-
lyzing a representative panel of liver-specific genes, all normally expressed in adult hepatocytes ( Fig. 3A-I, Supplemental Table 1). RNA levels were examined in HepaMN cells and compared with the differentiated human hepatoblastoma-derived HepaRG cell line. HepaMN cells expressed the albumin gene at a higher level than HepaRG, but expressed the other liver-specific genes at low or undetectable levels.

Global outlook by principal component analysis (PCA).
To clarify the differentiated state of HepaMN, we compared the gene expression levels in HepaMN cells and hepatocyte-differentiated ESCs using Agilent Technologies GeneChip oligonucleotide arrays.
LGR5XF32d30 and LGR5XF32d60 used in this study   www.nature.com/scientificreports/ are human ESCs that were differentiated into hepatocytes for 30 and 60 days, respectively. We then performed PCA from the expression data of liver-associated genes, i.e. developing and mature hepatocyte markers (Supplemental Table 2). PCA based on liver-associated genes demonstrated that HepaMN cells are grouped into the category that includes embryonic stem cell-derived terminally differentiated hepatocytes (circled in red and green in Fig. 3J,K, respectively). The genes on the PC1 axis, based on developing and mature hepatocyte markers, are listed with correlations in Table 1A,B, respectively. These similarities led us to hypothesize that HepaMN cells maintain a specific differentiated state that strongly resembles hepatocytes.

In vivo analysis of HepaMN cells. To address whether HepaMN cells exhibit hepatocytic phenotypes
in vivo, they were injected into subcutaneous tissue (1.0 to 5.0 × 10 7 cells/site) of immunodeficient SCID mice (Fig. 4A,B). Twenty independent experiments revealed that HepaMN cells reproducibly showed hepatocytic www.nature.com/scientificreports/ morphology with canalicular structure in vivo nine days after implantation (Fig. 4C,D). Mock surgery with injection of PBS alone did not show the presence of hepatocyte-like cells at the injection sites. Immunohistochemical analysis showed that HepaMN cells were positive for Hep1 (CPS1), albumin, and AE1/3, but negative for α-fetoprotein (AFP) (Fig. 4E-L). HepaMN cells were also immunohistochemically analyzed in the spleen seven days after implantation ( Fig. 4M-V). Again, HepaMN cells were positive for Hep1, albumin, and AE1/3. HepaMN cells also stained positive for MRP2 at the cell membrane and CK19 in the cytoplasm. Nor did HepaMN exhibit cell proliferation histopathologically and produce obvious tumors macroscopically in the subcutaneous tissue of the SCID mice during the monitoring period (> 30 days), implying that HepaMN cells do not have an oncogenic potential. We also performed immunohistochemistry using an antibody to Ki67 to investigate whether HepaMN cells have a proliferative capability in vitro and in vivo (Supplemental Fig. 2). HepaMN cells in culture are positive at 58.1% on average, but HepaMN cells at the implantation site are positive only at 6.4% on average. HepaMN cells dramatically decreased their proliferative capability after the implantation.
Cytochrome P450 (CYP) mRNA induction after drug treatment. In order to examine HepaMN cells for CYP induction, HepaMN cells were exposed to omeprazole, phenobarbital, or rifampicin (Fig. 5A). CYP1A1 and CYP1A2 were induced with exposure to omeprazole or β-naphthoflavone (Supplemental Fig. 3). CYP2B6 was not induced with exposure to phenobarbital. HepaMN cells were damaged or killed after exposure to phenobarbital, and thus the CYP2B6 induction was performed using RNA from the surviving cells. CYP3A4 and CYP1A2 were induced by exposure to rifampicin 8.5-and 2.1-fold on average, respectively, while CYP2B6 did not show any induction. Primary human hepatocytes were examined for CYP induction along with HepaMN cells (Supplemental Fig. 4). We further analyzed CYP3A4 metabolism enzyme activity quantitatively in HepaMN cells at sub-confluence and compared the results with the activity observed in HepaRG cells (Fig. 5B,C). CYP3A4 activity was 38.6 pmol/min/mg protein, and the CYP3A4 value was lower in HepaMN cells than in HepaRG cells. The CYP3A4 activity in HepaMN cells was specifically inhibited by the specific CYP3A4 inhibitor ketoconazole. Expression levels of albumin and AFP remained unchanged (Fig. 5D,E).

Discussion
To date, numerous human hepatocyte cell lines have been used for drug interaction studies and hepatitis virus infection. We herewith introduce a novel hepatocyte line, HepaMN, that was established from a patient with biliary atresia, a childhood disease of the liver in which bile ducts are abnormally narrow or absent. While HepaRG and HepG2 were generated from hepatoma or hepatocellular carcinoma, HepaMN cells were generated from untransformed hepatocytes, probably hepatic progenitors, like other epithelial progenitors. From this viewpoint, HepaMN cells are the first immortalized hepatocyte line with normal function and diploid chromosomes. It is most noteworthy that HepaMN cells exhibited comparable expression levels of the albumin gene with HepaRG, and exhibited liver-like morphology in vivo. Liver, a multifunctional organ, consists of hepatocytes, pericytes, and stellate cells. Among these cells, hepatocytes play a key role in metabolism and detoxification. However, freshly isolated hepatocytes show considerable variation from lot to lot and are difficult to propagate in vitro. With comparable physiological functions to freshly isolated hepatocytes, the cloned cell line HepaMN was selected from among 11 immortalized hepatocyte clones from Japanese samples (Supplemental Tables 3, 4).
In the general scheme of model-based prediction from the Food and Drug Administration (FDA) draft guidance, an in vitro induction system is established in cultured human hepatocytes from 3 donors (FDA, 2017). Despite the variability seen among hepatocyte lots, freshly isolated human hepatocytes have been the system of choice for evaluating enzyme induction in vitro. In addition to human hepatocytes, cell lines such as HepG2, Huh7, THLE-2, PLC-PRF-5, and AML-12 have been used for drug interaction studies. Likewise, HepaMN cells may serve another hepatocyte model cell because of its stable CYP3A4 induction and constitutive albumin gene expression. It is also noted that phenobarbital treatment did not cause a significant CYP2B6 induction in HepaMN cells. This may be explained by phenobarbital cytotoxicity or absence of the CAR-PXR cross-talk usually observed in primary hepatocytes [17][18][19] . Most importantly, HepaMN cells resembled normal hepatocytes with regards to two important hepatic functional features: (1) maintenance of efficient proliferation accompanied by constitutive expression of albumin, and (2) the ability to display metabolic functions. In addition to the vitro benefits, immunodeficient animals with human HepaMN cells in livers can also be prepared for in vivo hepatotoxicity tests, albeit the low implantation rate of HepaMN cells into the mouse liver through the spleen. In order to generate mice with humanized livers, more efficient implantation methods and routes need to be determined, i.e. cell injection through the portal vein.

Conclusions
Novel regenerative medicine products such as decellularized liver, liver assistance devices, and bioartificial livers have recently been developed. For pharmaceutical evaluation of these systems, HepaMN cells may be valuable as a cost-effective and relatively easy-to-use substitute for hepatocytes dissociated from livers or from primary culture. Most importantly, HepaMN cells possess the outstanding characteristics of immortality and retention of hepatic features even after a long culture period.   CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1, and CSII-TRE-Tight-CDK4 R24C was previously described 20 .

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).
Immunocytochemical analysis. 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 preincubated 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. Karyotypic  www.nature.com/scientificreports/ 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).
Quantitative RT-PCR. 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 analysis 23,24 . 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 detail 25  www.nature.com/scientificreports/ 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. HepaMN   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 tubes 27 . 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 5C 18 -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. Statistical analysis was performed using the unpaired two-tailed Student's t test.