Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival

Experimental Cell Research xxx (xxxx) xxx–xxx Contents lists available at ScienceDirect Experimental Cell Research journal homepage: www.elsevier.co...

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Experimental Cell Research xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Experimental Cell Research journal homepage: www.elsevier.com/locate/yexcr

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival Hua-Lian Hanga,1, Xin-Yu Liub,1, Hai-Tian Wangb, Ning Xua, Jian-Min Bianb, Jian-Jun Zhanga, ⁎ ⁎ Lei Xiaa, , Qiang Xiaa, a b

Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Immortalized cells Adult human hepatocytes Hepatocyte nuclear factor 4A Hepatic differentiation Chromosome instability Acute liver failure Liver function

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24 h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.

1. Introduction Orthotopic liver transplantation (OLT) improves the survival and quality of life of patients with end-stage liver diseases, but this procedure is expensive, complex, and limited by the rarity of donor livers [1]. Primary human hepatocytes constitute a suitable model for pharmacological and toxicological studies, cell transplantations, and bioartificial extracorporeal liver support systems [2]. Animal studies showed that transplantation of isolated hepatocytes could be useful in the treatment of patients with liver-based metabolic diseases and liver failure [3]. Hepatocyte transplantation (HT) could be used as a “bridging” procedure to buy time for patients awaiting liver transplantation [2]. In some cases, the transplanted cells could provide metabolic



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support long enough for the surviving host liver cells to regenerate, obviating the need for whole organ transplantation [2]. Unfortunately, the sources of primary human hepatocytes are limited and their isolation is time-consuming and expensive [2,4]. Yet, major obstacles to the broad clinical use of HT include the competition with OLT for the few suitable donor livers and the fact that primary hepatocytes cannot be readily expanded in vitro [2,5]. Immortalized human hepatocytes (IHH) generated by the introduction of immortalizing genes are a potential cell source for HT. Adult IHH [6,7] and fetal hepatocytes [8] have been generated by transduction of the simian virus (SV) 40 T or human telomerase reverse transcriptase (hTERT) gene. Most of these hepatocytes retain the characteristics of the primary hepatocytes and did not show

Corresponding authors. E-mail addresses: [email protected] (L. Xia), [email protected] (Q. Xia). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.yexcr.2017.08.020 Received 9 February 2017; Received in revised form 10 August 2017; Accepted 12 August 2017 0014-4827/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Hang, H.-L., Experimental Cell Research (2017), http://dx.doi.org/10.1016/j.yexcr.2017.08.020

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(NaCl 136.75 mM, KCl 5.37 mM, KH2PO4 0.441 mM, NaHCO3 4.17 mM, pH 7.3) for 10 min. Occlusion of the suprahepatic inferior vena cava was performed before the tissue was perfused with 500 ml of a pre-warmed (37 °C) digestion buffer containing 0.05% collagenase IV (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) for 20–30 min. After digestion, the cell suspension was obtained by smooth mechanic dissociation, filtered through a 100-µm stainless steel strainers, and centrifuged three times at 50 × g for 5 min at 4 °C to remove cell debris. Hepatocyte viability was measured by the trypan blue exclusion test. Hepatocytes were seeded on 24-well fibronectin-coated monolayer culture plates at 2 × 105 cells per well in HepatoZYME-SFM medium (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) containing hepatocyte growth factor (25 ng/ml), penicillin (50 U/ml), streptomycin (50 µg/ ml), and L-glutamine (2 mM) at 37 °C in 95% O2/5%CO2. The medium was changed every 24 h.

tumorigenicity when implanted subcutaneously in nude mice [9–11], but some immortalized human epithelial cells transduced with SV40T showed persistent chromosome instability with passages [12,13] and TERT-driven cell proliferation was still not genoprotective because it was associated with activation of the c-myc oncogene [14]. Therefore, the risk of acquiring tumorigenicity by long-term passage could not be eliminated. To address these issues, we have previously shown that primary human hepatocytes can be expanded in vitro through transfection of lentiviral vectors coding for hTERT [15]. The resulting cell line showed appropriate functionality and hepatic differentiation. Nevertheless, tumorigenicity could remain an issue. Hepatocyte nuclear factors (HNFs) have been shown to play important roles in liver development and hepatocyte differentiation. Maintenance of hepatocyte differentiation and control of liver-specific gene expression are attributed in large part to HNFs [16,17]. This class of proteins includes five families of transcriptional regulators: HNF1, HNF3, C/EBP, HNF4, and HNF6. In particular, the nuclear receptor HNF4 is a key regulator of both hepatocyte differentiations during embryonic development and maintenance of a differentiated phenotype in adult [18]. It has been shown that HNF4A is essential for morphological and functional differentiation of hepatocytes, whereas the expression of HNF1A is not an absolute requirement for mammalian liver development [18]. HNF4A is the dominant regulator of the epithelial phenotype of hepatocytes and is essential for normal liver architecture [19]. Forty-two percent of the genes occupied by RNA polymerase II in hepatocytes are bound by HNF4A [20]. Mice lacking hepatic expression of HNF4A showed that HNF4A regulates the expression of many target genes [21,22]. HNF4A, as an orphan nuclear receptor, activates gene transcription in the absence of exogenous ligand [23,24]; therefore, unlike classic nuclear receptors, the transcriptional activity of HNF4A is largely dependent on the selective interaction of tissue-specific or independently regulated coregulators with its activation function 2 (AF2) domain to stimulate target genes in a tissue-specific and metabolically regulated gene-specific manner [25]. Nevertheless, it remains unknown whether HNF4A affects the generation of IHH and the outcomes of long-term maintenance of these cells. Having established suitable pre-incubation conditions [26] and a microencapsulated cryopreservation method [27] for human hepatocytes, we established a human hepatocyte bank through the isolation of primary human hepatocytes after partial hepatectomy. Using these cells, we examined whether HNF4A expression efficiently induced hepatic differentiation of IHH and investigated the underlying mechanisms. To assess whether the IHH met the requirement for clinical applications, their liver functions and differentiation were compared with those of primary human hepatocytes.

2.2. Establishment of immortalized human hepatocytes by lentivirusmediated hTERT overexpression in primary hepatocytes The immortalizing gene hTERT was amplified from the retrovirus vector pBabe puro hTERT (kindly provided by Professor Wright from the University of Texas Southwestern Medical Center, USA) as previously published [30], confirmed by sequencing, and inserted into the lentiviral expression vector pCC-FU-3FLAG. High-titer recombinant virus stocks of the HIV-derived lentivectors pGC-FU-hTERT-3FLAG, pHelper 1.0, and pHelper 2.0 (Genechem Co., Ltd, Shanghai, China) were cotransfected into 293T cells and packaged using the lentivector expression system (Genechem, Shanghai, China). Lentivirus-mediated TERT overexpression vectors were prepared and titered to 5 × 108 TU/ ml (transfection units). After 2 days of culture, primary hepatocytes were transduced with hTERT lentiviral vectors at a multiplicity of infection (MOI) of 30 in the presence of 5 µg/ml of polybrene (Sigma, St Louis, MO, USA) at 37 °C for 12 h. IHH were trypsinized 2 weeks later, further grown for 2 weeks in hepatocyte medium, and subsequently expanded in HepatoZYMESFM medium (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) containing hepatocyte growth factor (25 ng/ml), penicillin (50 U/ml), streptomycin (50 µg/ml), and L-glutamine (2 mM). These IHH were cloned in 96-well plates by limiting dilution. Hepatocyte markers and tumorigenicity (c-myc) of IHH were assessed by RT-PCR and immunocytochemistry of cytokeratin 18. The human hepatocellular carcinoma cell line HepG2 was used as positive control. 2.3. RT-PCR Total RNA was extracted from IHH and primary hepatocytes using Trizol (GIBCO, Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer's instructions. RNA concentration was determined at 260 nm using a photometer (Eppendorf, Netheler-Hinz, Hamburg, Germany). RNA purity was determined by the 260/280 nm absorbance ratio. Total RNA was treated with DNase. First strand complementary DNA (cDNA) was synthesized by a reverse transcription system (Promega, Madison, WI, USA). All primers were synthesized by Invitrogen (Shanghai, China). The primer sequences and PCR reaction conditions are shown in the Supplementary Table 1. PCR was performed in a Supermix DNA thermal cycler (GIBCO, Invitrogen Inc., Carlsbad, CA, USA). GAPDH was used as the internal standard. The PCR products were analyzed by 2% agarose gel.

2. Material and methods 2.1. Hepatocyte isolation and culture Adult human liver specimens were collected from surgeries for benign diseases (hepatic hemangioma and donor livers). Livers with severe infectious diseases (hepatitis B and C, or HIV) and tissues from previously histologically proven highly cirrhotic or highly steatotic livers, as well as livers from patients with GGT levels > 60 U/l [28] were excluded. The study was approved by the Ethics Committee of the Affiliated Ren Ji Hospital of Shanghai Jiao Tong University, China. Written consent for the use of the samples for research purposes was obtained from all patients. The methods were carried out in accordance with the approved guidelines. Hepatocyte isolation was performed under sterile conditions using a modified extracorporeal collagenase perfusion technique [26] based on a published protocol [29]. Briefly, the liver samples were cannulated, ligated, and immobilized through the branches of the partial hepatic artery and vein as soon as the liver was removed from the patient. The partial livers were flushed with 2000 ml of D-Hanks washing buffer

2.4. Telomerase activity Telomerase activity in IHH was assayed by the PCR-based telomeric repeat amplification protocol (TRAP) using a TRAPEZE telomerase detection kit (Chemicon, Temecula, CA, USA), according to the manufacturer's protocol. Telomerase extracts from IHH were diluted to 100 ng/ml. Two μl of each telomerase extract was subjected to the 2

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complementary pair of oligonucleotides for a sense-only control RNA was designed: 5'-gat ccG TGT GTG ACT TGC TGT GGT CTT CAA GAG ATT TTT Tg-3' and 5'-aat tcA AAA AAT CTC TTG AAG ACC ACA GCA AGT CAC ACA Cg-3'. The oligonucleotide pairs containing BamHI and EcoRI overhangs were annealed and ligated to a linearized RNAi-Ready pSIREN-RetroQZsGreen vector digested with BamHI and EcoRI (Clontech Laboratories Inc., Mountain View, CA, USA). The resultant constructs were amplified, purified, and sequenced. For production of high-titer retrovirus vectors expressing HNF4A small hairpin RNA, EcoPack-293 cells (Clontech Laboratories Inc., Mountain View, CA, USA) were plated at 50–75% confluence and the recombinant vectors were transfected by a modified calcium phosphate method. After incubation at 37 °C for 2 days, the supernatant containing the viral vector was collected and filtered. Human hepatocytes were infected with the retroviral vector in the presence of polybrene (5–10 µg/ml) and incubated for 24 h. The supernatant was removed and replaced with normal growth medium. Cells were grown for 48–72 h and assessed by fluorescence microscopy (Olympus, Tokyo, Japan). Infected populations exhibiting 60–75% of green fluorescent cells were used for the subsequent experiments. HNF4A knockdown was confirmed by RT-PCR and western blot.

TRAP assay before being loaded on a 10% polyacrylamide gel. The products were visualized by SYBR Green I staining (Invitrogen Inc., Carlsbad, CA, USA). 2.5. MTT assay Cell growth curves were determined by the MTT assay. Cells in the exponential growth phase were seeded into a 96-well plate (4 × 103 cells/well) and cultured overnight. MTT (Sigma, St Louis, MO, USA) (20 μl, 5 mg/ml) was added to each well, and the plate was incubated at 37 °C for 4 h. Formazan crystals were dissolved in DMSO. Absorbance was determined with a microplate reader (Bio-Tek, Winooski, VT, USA) at 570 nm. 2.6. Cell cycle Cell cycle was determined using the Cycle TEST PLUS DNA kit (BD Biosciences, Franklin Lake, NJ, USA), according to the manufacturer's protocol, and a FACScan (BD Biosciences, Franklin Lake, NJ, USA). 2.7. Periodic acid-Schiff staining

2.11. Real-time RT-PCR

The periodic acid-Schiff (PAS) method was used to examine hepatocytes for glycogen storage. The adherent cells were oxidized in 1% periodic acid for 10 min, treated with Schiff's reagent for 15 min, counterstained with hematoxylin for 1 min, and assessed under a CX40RF310 light microscope (Olympus, Tokyo, Japan).

Total RNA was extracted with TRIzol (Invitrogen Inc., Carlsbad, CA, USA) and reverse transcripted using Superscript II (Invitrogen Inc., Carlsbad, CA, USA). Quantitative PCR was carried out using the SYBRgreen qPCR kit (Takara Bio, Otsu, Japan), gene specific primers, as shown in Supplementary Table 2, and a Mx7300P Real-Time PCR System (Stratagene, Wilmington, DE, USA). Gene expression was quantified by the 2-ΔΔCt method [31], using β-actin as an internal standard.

2.8. Urea production Cells were incubated in the presence of 5 mM ammonium chloride for 24 h before the concentration of secreted urea in the culture media was determined using a urea assay kit (Biochain Institute Inc., Hayward, CA, USA). The absorbance was measured at 520 nm with a Cytofluor microplate reader (Benchmark, Hercules, CA, USA).

2.12. Isolation of nuclear and cytoplasmic proteins Cytoplasmic and nuclear proteins were isolated from IHH cells using the Nuclear and Cytoplasmic Proteins Extraction Kit (Pierce, Thermo Pierce, Rockford, IL, USA), according to the manufacturer's instructions.

2.9. HNF4A overexpression HNF4A cDNA generated by RT-PCR from total RNA prepared from primary hepatocytes yielded a 1.4 kb product. The primers were: HNF4A forward 5'-AGG ATC CAT GCG ACT CTC CAA AAC CCT CG-3' and reverse 5'-AGA ATT CCC TAG ATA ACT TCC TGC TTG GTG-3'. The PCR product was separated on 0.9% agarose and the 1.4-kb fragment was purified with a commercial purification kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China). The fragment was inserted into the pcDNA3.1 vector (Invitrogen Inc., Carlsbad, CA, USA) and sequenced commercially (Invitrogen Inc., Carlsbad, CA, USA). Transient transfection of 5 × 105 cells into a single clone of HNF4A negative IHH was performed using Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, USA), according to the manufacturer's instructions, with either 4 µg of pcDNA3.1-HNF4A or 4 µg of empty pcDNA3 vector plasmids as vector control. G418 was used for 2 weeks to screen for stably transduced cells. The successful transfection of the HNF4A was confirmed by RTPCR and western blot.

2.13. Western blot Total proteins were extracted using a protein extraction reagent (Applygen Technologies Inc., Beijing, China), according to the manufacturer's instructions. Protein concentration was determined by a commercial kit (KeyGen Biotech Co., Beijing, China). Equal amounts of proteins were separated by 10% SDS-PAGE and transferred to polyvinyl membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% milk powder in TBS with 0.05% Tween 20 (TBST). Proteins were detected by western blot using rabbit anti-human CYP3A4 polyclonal antibody (Millipore corp., Billerica, MA, USA), goat anti-human ALB antibody (Bethyl Laboratories, Montgomery, TX, USA), mouse antihuman HNF4A antibody (R & D Systems, Minneapolis, MN, USA), rabbit anti-human beta-catenin antibody (Abcam, Cambridge, MA, USA; ab6302), rabbit anti-human histone H2A antibody (Abcam, Cambridge, MA, USA; ab88770), β-actin (Sigma, St Louis, MO, USA). Following overnight incubation with the primary antibody, each blot was washed four times with TBST buffer. Blots were incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were detected using an enhanced chemiluminescence reagent (Pierce, Rockford, IL, USA).

2.10. HNF4A knockdown A retrovirus vector-based short hairpin RNA (shRNA) was prepared using the BD Knockout RNAi system (Clontech Laboratories Inc., Mountain View, CA, USA) and used to generate HNF4A-knockdown primary hepatocytes. The PAGE-purified complementary oligonucleotide pair for the shRNA was synthesized to target the coding region of human HNF4A catalytic subunit as follows: 5'-GAT CCG TGA CAC GTC CCC ATC AGA ATA GTG CTC CTG GTT GTT CTG ATG GGG ACG TGT CAC TTT TTT G-3' and 5'-AAT TCA AAA AAG TGA CAC GTC CCC ATC AGA ACA ACC AGG AGC ACT ATT CTG ATG GGG ACG TGT CAC G-3' A

2.14. Karyotype analysis Karyotype analysis was carried out by G-band pattern staining of primary hepatocytes [32]. Chromosomes of a minimum of 50 3

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2.20. Electrophoretic mobility shift assay of β-catenin

metaphase cells were counted, and 10 cells were karyotyped for each cell line.

Nuclear proteins were extract from the IHH. A 10-μg nuclear protein aliquot was incubated in binding buffer with a biotin-labeled β-catenin consensus oligonucleotide probe (5’-AAG AGG CCC CCA CTT CAA AGG TGT GAG AAG AGC-3’) for 30 min. The specificity of the DNA/protein binding was determined by adding a 100-fold molar excess of unlabeled β-catenin oligonucleotide for competitive binding 10 min before adding the biotin-labeled probe.

2.15. Encapsulation of immortalized human hepatocytes Encapsulation was performed as previously described [27]. IHH (2 ml, 1 × 106 cells/ml) were diluted with 2 ml of sterile filtered 4% alginate (Hushi, Shanghai, China) dissolved in Ca2+-free DMEM (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA). The suspension was extruded using a droplet generator NISCO encapsulator (NISCO, Zurich, Switzerland) and sprayed into a 100 mmol/l CaCl2 solution (Qianchen, Shanghai, China). The microcapsules were washed with normal saline solution and treated with 1% poly-L-lysine for 5 min. The beads were immersed in a 0.15% alginate solution for 10 min and washed again. The beads were incubated in 55 mmol/l sodium citrate solution (Sigma, St Louis, MO, USA) for 10 min to dissolve the alginate core. Microcapsules had a diameter of 250–350 µm and contained 60–80 cells.

2.21. Statistical analysis Data were provided as mean ± standard error of the mean (SEM). Statistical significance was evaluated by one-way analysis of variance (ANOVA) with the SNK post hoc test or Student t-test. Survival was analyzed using the Kaplan-Meier method, and differences were evaluated using the log-rank test. Statistical analysis was performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Two-sided P-values < 0.05 were considered statistically significant.

2.16. Rat model of acute liver failure and transplantation of encapsulated immortalized human hepatocytes

3. Results 3.1. Establishment of immortalized human hepatocytes

Male Sprague-Dawley rats (eight-week-old, weighting 300–350 g) were housed at 20–23 °C under 12-h light/12-h dark cycles. The rats had free access to food and water. The rats were injected intraperitoneally with 1.0 g/kg of D-galactosamine (Sigma, St Louis, MO, USA) to induce ALF [33]. Transplantation of encapsulated IHH was performed 24 h after ALF modeling. The rats were randomly divided into three groups. In the empty capsules group (n = 6), 2 ml of empty capsules were injected into the peritoneal cavity. In the encapsulated IHH group (n = 6), 5 × 107 encapsulated normal IHH (2 ml) were injected into the peritoneal cavity. In the encapsulated IHH with overexpression of HNF4 group (n = 6), 5 × 107 encapsulated IHH with overexpression of HNF4 (2 ml) were injected into the peritoneal cavity (n = 6). The animals were monitored for 7 days. All procedures and animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

The viability and plating efficiency of hepatocytes after partial hepatectomy using the two-step extracorporeal collagenase perfusion technique were 75.0 ± 4.6% and 72.0 ± 6.0%, respectively [26]. After 2 days of culture, hepatocytes were lentivirally transduced with the hTERT gene at a MOI of 30. The mRNA expression of hTERT is shown in Fig. 1A. Telomerase activity was sustained in these lines at late passages (200 PDs) (Fig. 1B). We observed extended life spans of cells exhibiting hepatocyte morphology comparable to primary human hepatocytes. The cells exhibited epithelial-like morphology throughout the culture periods, as shown in Fig. 2A. The IHH proliferated for over 200 PD without growth arrest, with an average doubling time of 60 h (2–3

2.17. Biochemistry and survival Blood samples from the caudal vein were taken daily and analyzed with an automatic analyzer (7600, Hitachi, Tokyo, Japan) for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin levels. Survival was evaluated for 7 days. 2.18. Histopathology The animals were sacrificed by cervical dislocation. Liver specimens were collected, fixed by 4% paraformaldehyde, embedded in paraffin, sectioned at 4 µm sections, and stained with hematoxylin and eosin (H & E). 2.19. Dual-luciferase reporter assay To detect the effect of HNF4α on β-catenin/Tcf activity, HepG2 or 293T cells were co-transfected with the pTcf-Luc reporter plasmid and the internal control pRL-TK (Promega, Madison, WI, USA). At 12 h posttransfection, the cells were transfected with pcDNA3.1-HNF4A or empty pcDNA3 vector plasmids as vector control. After 24 h, the cells were harvested and the luciferase activity was measured using the dualluciferase reporter assay system (Promega, Madison, WI, USA), according to the manufacturer's instructions.

Fig. 1. mRNA expression and activity of hTERT detected in immortalized human hepatocytes (IHH) established by lentivirus-mediated hTERT overexpression in primary human hepatocytes (P). (A) hTERT mRNA expression was determined by RTPCR. GAPDH was used as inner control. M: DNA marker. (B) Telomerase activity was examined by TRAP assay and sustained in IHH at late passage (200 population doubling (PD)) (P-, IHH-, and C-: 80 °C heat-inactivated control of P, IHH, and C. C: hTERT positive control; IC: internal control to normalize the efficiency of PCR amplification; C°: contamination control).

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Fig. 2. Characterization of IHH established by lentivirus-mediated hTERT overexpression in primary human hepatocytes. (A) Phase contrast microscopy photographs of freshly isolated primary human hepatocytes (P) 2 days after primary culture and morphology of IHH at 200 PD. The IHH exhibited epithelial-like morphology throughout the culture periods (magnification: × 100). (B) Comparative growth curves of P and IHH determined by MTT assay. (C) Cell cycle distributions of P and IHH were analyzed by flow cytometry. (D) Immunocytochemistry for mature human hepatocytes expressing the liver specific markers CK18 in the membrane (magnification: × 100). Data are shown as mean ± standard error of the mean (SEM) from three independent experiments performed in triplicate. *P < 0.05 vs. primary hepatocytes.

immortalized human hepatocytes were positive for ALB, CYP3A4, CEBPE, HNF1A, and HNF4A mRNA expressions (Fig. 3A), exhibiting a similar profile as the primary cultured human hepatocytes. ALB, Cytochrome P450 3A4 (CYP3A4), and HNF4A protein expression was also detected in both the immortalized and primary hepatocytes (Fig. 3B).

days) (Fig. 2B). The cell cycle distributions of primary hepatocytes and IHH were analyzed by flow cytometry. As shown in Fig. 2C, among primary hepatocytes, 82.5 ± 6.1% were in G0/G1 and 17.5 ± 6.1 were in S+G2/M, compared with IHH, among which 66.4 ± 5.5% were in G0/G1 and 33.6 ± 5.5% were in S+G2/M. Furthermore, we confirmed by immunocytochemistry that IHH expressed the liver-specific marker CK18, as shown in Fig. 2D. To establish an IHH line with proper functions and without tumorigenicity, the functional gene expressions of 24 clones after limiting dilution were assessed by RT-PCR. RNA was extracted and characterized for livers specific genes including albumin (ALB), cytochrome P450 2B6 (CYP2B6), cytochrome P450 (CYP3A4), cytochrome P450 (CYP7A1), CCAAT/enhancer-binding protein epsilon (CEBPE), and asialoglycoprotein receptor (AGPR). In addition, RT-PCR was used to investigate the expression of hepatocyte nuclear factor 1α (HNF1A), hepatocyte nuclear factor 4α (HNF4A), hepatocyte nuclear factor 1β (HNF1B), and hepatocyte nuclear factor 4γ (HNF4G) (which are advanced functional transcription factors in the liver [31]) in every clone. The expression of the c-myc oncogene was also analyzed for evaluating the tumorigenicity of the cells. Based on the gene expression profile (Supplementary Table 3), four HNF4A-positive clones (no. 8, 9, 19, and 20) were selected for the establishment of IHH. To analyze whether HNF4A-positive IHH retained hepatocyte characteristics, we examined the expression levels of hepatocyte-enriched genes by RT-PCR and western blot. The results indicated that the

3.2. Effects of HNF4A overexpression on the hepatic function and differentiation markers, cell morphology, and functional analysis in the HNF4A-negative immortalized hepatocytes To examine the effects of HNF4A overexpression, we transfected the pcDNA3.1-HNF4A construct into HNF4A-negative IHH. An empty vector was used as negative control. A number of stable HNF4A transfectant clones with HNF4A overexpression were selected, as determined by RT-PCR and western blot. The HNF4A mRNA expression increased gradually and peaked on day 2, but gradually decreased over time after transfection (Fig. 4A). Western blot showed that the HNF4A protein expression in the HNF4A-negative IHH overexpressing HNF4A was higher than that in the HNF4A-negative IHH transfected with empty vector (Fig. 4G). This observation demonstrated that HNF4Anegative IHH can stably overexpress HNF4A. Hepatic function and differentiation of the cells were investigated by measuring the mRNA levels of hepatocyte-specific genes at 0, 1, 2, 3, and 4 days post-transfection. Expression of ALB mRNA was gradually increased during the four days and significantly increased at 2 and 4 5

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Fig. 3. Hepatocyte markers and tumorigenicity (c-myc) of IHH. (A) Human liver enriched markers and tumorigenicity (c-myc) were analyzed by RT-PCR. GAPDH was used as inner control. (B) Albumin (ALB), cytochrome P450 3A4 (CYP3A4), and hepatocyte nuclear factor 4 (HNF4A) were determined by western blot. β-actin was used as an inner controls.

by PAS staining was also increased in HNF4A-overexpressing cells, while negative control did not (Fig. 5C).

days (Fig. 4B). ALB mRNA expression was decreased after 4 days (data not shown). The mRNA expression of CYP3A4 and CEBPE was similar to that of ALB and was significantly increased on days 1 and 2 after transfection (P < 0.05) (Fig. 4C and D). Expression of α1-antitrypsin reached a plateau on day 1 and decreased gradually (Fig. 4E). Transferrin mRNA also increased on day 0 and reached a peak on days 1 and 2 (Fig. 4F). Western blot was performed for the expression of hepatocyte-specific proteins 2 days after HNF4A overexpression. The results showed that ALB production and CYP3A4 induction in the cells with HNF4A overexpression were increased compared with those without overexpression (negative control), and they showed nearly as much expression as primary human hepatocytes (positive control) (Fig. 4G). The stable HNF4A-expressing hepatocytes exhibited morphology changes after 2 weeks of G418 selection: the cells became extended and enlarged (Fig. 5A). We next examined whether HNF4A-negative IHH overexpressing HNF4A exhibited increased urea synthesis. Urea levels were increased by overexpression of HNF4A, as measured 24 h after ammonium chloride addition, similar to that of primary human hepatocytes (P > 0.05) (Fig. 5B). Hepatic glycogen accumulation evaluated

3.3. Effects of HNF4A knockdown on cell morphology, hepatocyte markers, and karyotype analysis in primary human hepatocytes Two pairs of oligonucleotides encoding HNF4A-specific shRNAs were designed using retrovirus-mediated expression to silence HNF4A expression. After 48 h of transfection, HNF4A levels were decreased effectively in primary hepatocytes by HNF4A shRNA treatment (Fig. 6A). We also examined changes in HNF4A protein in primary human hepatocytes, which normally express high levels of the HNF4A protein. The HNF4A shRNA markedly reduced HNF4A protein levels as compared with the controls (primary human hepatocytes and primary human hepatocytes transfected with scramble shRNA), but HNF4A protein levels in the scramble shRNA group were similar to that observed in the primary human hepatocytes (Fig. 6B). As compared with the two controls, the function (ALB and CYP3A4) and differentiation-related gene (transferrin, α1-antitrypsin) expression 6

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Fig. 4. Effects of HNF4A overexpression on hepatocyte markers in HNF4A-negative IHH. The HNF4A-negative IHH overexpressing HNF4A (IHHHNF4A(+)) after 0, 1, 2, 3, and 4 days. (A) HNF4A, (B) ALB, (C) CYP3A4, (D) CEBPE, (E) a1-antitrypsin (AAT), (F) transferrin mRNA expressions were measured by real-time RT-PCR. β-actin was used as an inner control. HNF4A-negative IHH stably transfected with emptor vector [IHHHNF4A(-)] and primary hepatocytes (P) groups acted as negative and positive controls, respectively. Data are shown as the relative mRNA levels to those of the IHHHNF4A(-) group. (G) Protein expressions of HNF4A, albumin, CYP3A4, AAT, and transferrin in IHHHNF4A(-) treated with overexpression of HNF4A gene (IHHHNF4A(+)) after 2 days were examined by western blot. β-actin was used as inner control. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate. *P < 0.05, **P < 0.01 vs. IHHHNF4A(-); #P < 0.05, ##P < 0.01 vs. primary hepatocytes.

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Fig. 5. Effects of HNF4A overexpression on cell morphology and functional analysis in HNF4A2-negative IHH. (A) The morphology of stable HNF4A overexpression HNF4Anegative IHH (IHHHNF4A(+)) and HNF4A-negative IHH transfected with emptor vector (IHHHNF4A(-)) were observed by phase contrast microscopy (× 40, × 100). (B) Urea production. Cells were stably transfected with PCDNA3.1-HNF4A overexpression for 3 days. Cells were incubated in the presence of 5 mM ammonium chloride for 24 h before the concentration of secreted urea in the culture media was determined by colorimetry. Data are expressed as the mean ± SEM of three experiments performed in triplicate. *P < 0.05, **P < 0.01 vs. IHHHNF4A(-). (C) Periodic acid-Schiff (PAS) staining of IHH overexpressing HNF4A after 3 days. Glycogen stored in the cells was stained via PAS staining (× 100). The arrows indicate PASpositive cells. Emptor-vector (PCDNA3.1+) transfected IHH cultured in medium (IHHHNF4A(-)) were used as a negative control. Primary human hepatocytes were used as a positive control.

in both cell types (Fig. 7C). To confirm our result, the impact on βcatenin activation by HNF4α was evaluated by EMSA after overexpression of HNF4α. Again, HNF4α overexpression could significantly promote β-catenin activation (Fig. 7D).

levels of hepatocytes were not obviously changed. As c-myc is an important factor in tumorigenicity of hTERT-mediated immortalization, we examined changes in c-myc after knockdown of HNF4A in primary human hepatocytes, and surprisingly found that c-myc was significantly elevated in hepatocytes treated with HNF4A shRNA (Fig. 6C). In order to investigate changes in hepatocytes phenotype after HNF4A shRNA, hepatocytes were cultured for 2 weeks after retrovirus transfection and karyotype analysis was performed. Chromosomal abnormalities were observed in hepatocytes transfected with HNF4A shRNA. We prepared chromosome spreads 5 days after primary human hepatocytes were depleted for HNF4A and determined the number of chromosomes per spread, scoring cells as diploid (2n = 46) or aneuploid (30 < 2 n < 60, 2n ≠ 46). Pronounced whole-chromosome aneuploidy was observed after depletion of HNF4A and the number of metaphases in these cells showing abnormalities. When one interchange was counted as one break [34], the total number of aneuploidy was approximately four fold greater than that in the scramble shRNA transfected cells (P < 0.01) (Fig. 6D).

3.5. Transplantation of encapsulated IHH overexpressing HNF4A could improve liver function, survival, and histopathology in a rat model of acute liver failure The encapsulated beads were round in shape and identical in size with a mean diameter of 600–800 µm, each containing about 1000 cells (Fig. 8A and B). To determine the effect of transplantation of encapsulated IHH into rats with ALF, we measured the serum levels of ALT, AST, and total bilirubin. In the empty capsule group, ALT levels peaked at 4500 ± 175.78 IU/L 48 h after ALF modeling, with most of mortality occurring within 72 h. In the IHH and HNF4 overexpressing IHH groups, the ALT levels peaked at 1121.3 ± 129.94 IU/L and 75.57 ± 10.03 IU/L, respectively, but at 72 h instead of 48 h. In addition, the ALT levels of the HNF4-overexpressing IHH group were significantly lower than in the two other groups (all P < 0.05) (Fig. 8C). Similar patterns were observed for AST levels (Fig. 8D) and total bilirubin levels (all P < 0.05) (Fig. 8E). The survival rate for each group is shown in Fig. 8F. No rat died in the first 24 h after ALF modeling. The 72-h survival rates were 5%, 95%, and 100% among rats with empty capsules, encapsulated IHH, and encapsulated IHH overexpressing HNF4A, respectively. The 120-h survival rate was higher in the HNF4A overexpressing IHH group than that of the two other groups. The 7-day survival rate was 15% in the encapsulated IHH overexpressing HNF4A group, 15% in the encapsulated IHH group, and 5% in the empty capsule group. The rats in both IHH groups survived longer compared with the empty capsule

3.4. HNF4α promotes nuclear translocation of β-catenin in immortalized hepatocytes A study showed that the main indicator of Wnt/β-catenin signaling activation was β-catenin nuclear translocation [35]. In IHH, we detected a high endonuclear expression of β-catenin, but after transfection with the HNF4α plasmid, the nuclear translocation of β-catenin was promoted (Fig. 7A), while interference of HNF4α resulted in the opposite (Fig. 7B). We then performed dual-luciferase reporter assays to explore whether HNF4α overexpression could promote β-catenin/TCF activation in 293T and HepG2 cells. Luciferase assays showed that HNF4α significantly enhanced the activation of β-catenin/TCF reporter 8

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Fig. 6. Effects of HNF4A knockdown on cell morphology, hepatocyte markers, and karyotype analysis in primary human hepatocytes. Primary human hepatocytes were transduced with retrovirus-mediated HNF4A shRNA or scramble shRNA. (A) HNF4A mRNA and (B) HNF4A protein expressions were determined by real-time RT-PCR and western blot, respectively. (C) The mRNA expression of hepatocytes function and differentiation-related genes and tumorigenicity (c-myc) in P, HNF4A shRNA, and scramble shRNA were analyzed by RT-PCR. GAPDH was used as an inner control. (D) Karyotype analysis was carried out by G-band pattern staining. Chromosome instability occurred in hepatocytes transfected with HNF4A shRNA. Quantification of aneuploid cells from HNF4A shRNA and scramble shRNA groups. Data are expressed as the mean ± SEM of three experiments performed in triplicate. *P < 0.05, **P < 0.01 vs. scramble shRNA.

tumorigenicity test also suggest that IHH could express human hepatocyte antigens and glycogen. To establish an IHH line with hepatic function and lack of tumorigenicity, the functional gene expression of hepatic markers in 24 clones after limiting dilution was assessed by RTPCR. Four of the 24 selected clones were chosen on the basis of results from functional gene expression profiles assessed by RT-PCR. These four clones exhibited higher gene expression activities, relating mainly to synthesis and metabolism. All the other clones that were HNF4A negative appeared c-myc positive. CK18 is a liver-specific marker and is a cytoskeletal protein that is expressed in mature hepatocytes. We confirmed by immunocytochemistry that CK18 was expressed in the membrane of both immortalized and primary human hepatocytes. These results were further supported by the uninterrupted expression of HNF4A, a major transcription factor, that is required for hepatocyte differentiation and liver gene expression, as previously shown [8,18]. The results of the present study are in agreement with a report by Totsugawa et al. [7] that showed that reverted IHH (16-T3 cells) had increased expression of hepatic markers in association with enhanced levels of transcriptional factors C/EBPα and HNF-4α, resulting in higher albumin production and lidocaine metabolism. Recently, HNF4A was determined to be the key regulator of morphological and functional differentiation of hepatocytes, essential for

group (both P < 0.05). Histopathology of liver tissues demonstrated massive hemorrhage and extensive hepatocyte necrosis after ALF modeling (Fig. 8G). Hepatocyte necrosis area became smaller 24 h after encapsulated IHH transplantation (Fig. 8H). In addition, only mild hemorrhage and hepatocyte necrosis were observed in the portal areas after transplantation of encapsulated IHH overexpressing HNF4α (Fig. 8I) 4. Discussion IHH could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application [2]. This study aimed to determine the role of HNF4A in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with ALF. Results showed that HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. The newly established IHH reported in the present study revealed morphologic characteristics of mature hepatocytes in culture systems and expressed many liver markers such as ALB, CYP3A4, CEBPE, HNF1A1, and HNF4A. Immunohistochemistry, PAS staining, and 9

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Fig. 7. HNF4A influenced β-catenin nuclear translocation. (A) After HNF4A overexpression, promoted endonuclear expression of β-catenin was observed, normalized to histone H2A. (B) HNF4A deletion reduced β-catenin nuclear translocation. (C) 293T and HepG2 cell were transiently transfected with a β-catenin/TCF luciferase reporter construct with increasing amounts of HNF4A. Twenty-four hours after transfection, luciferase reporter assays were carried out. *P < 0.05. (D) HEK293 cells were transfected with emptor vector or HNF4A overexpression and treated as above. Lysates were used for EMSA using a radio-labeled β-catenin probe.

tumorigenic tendency. The results also support that cytogenetic change caused by the accumulation of chromosomal aberrations may be related to the dedifferentiation of IHH long-term passage [39]. Therefore, immortalized human liver cell line can be established by HNF4A positive expression screening. Although many studies reported that IHH did not show tumorigenicity in subcutaneous transplantation to SCID mice [6,8–10,13,15,39–41], the possibility of acquiring tumorigenicity after transplantation cannot be eliminated. Owing to safety concerns, we cannot advocate the transplantation of immortalized cells for clinical therapeutic purposes for now, but our results warrant efforts to continue to explore the potential of IHH for extracorporeal BAL devices. To test whether the transplantation of IHH with HNF4α overexpression could enhance the ability of the hepatocytes to alleviate ALF in vivo, we successfully encapsulated cells in alginate-poly-L-lysine alginate-coated beads, which form a barrier that protect the transplanted cells. The results showed that transplantation of IHH overexpressing HNF4a could significantly improve biochemical indicators and increase survival of rat models of ALF. Studies have shown that upregulated HNF4α expression could promote hepatocyte differentiation and enhance hepatocyte function [18,42,43]. The Wnt/β-catenin pathway plays an established role in embryogenesis and carcinogenesis [44,45], but its role in liver differentiation and function has not been deeply investigated. Previous studies have shown that Wnt/β-catenin signaling is crucial for hepatocyte differentiation [46–48]. HNF4α can inhibit the development of hepatocellular carcinoma via the Wnt/β-catenin signaling pathway [49,50]. In the present study, overexpression of HNF4α promoted β-catenin activation, indicating that the expression of HNF4α plays a positive role in the activation of the Wnt/β-catenin pathway. However, the specific mechanism need to be further studied. In conclusion, HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF. The regulation of hepatocyte function and differentiation by HNF4α might involve the Wnt/βcatenin signaling pathway partially. Future studies are needed to elucidate the mechanisms of HNF4α regulation of IHH differentiation and

metabolic function and the formation of a polarized hepatic epithelium as well as for cell-cell contact [19]. There are conflicting reports on the roles of HNF4A in liver carcinogenesis [36,37]. Nonetheless, the study by Lazarevich et al. [38] demonstrated that the loss of HNF4A expression is an important determinant for hepatocellular carcinomas (HCC) progression in mice and the development of an aggressive tumor phenotype. HNF4A expression is abrogated in highly invasive fast growing undifferentiated tumors. Reexpression of HNF4A in fast growing HCCs reversed the progressive phenotype and induced transition to an epithelial phenotype with expression of all major liverspecific transcription factors and genes. In the present study, to further confirm the role of HNF4A on hepatic function and differentiation of human hepatocytes, we examined the effect of HNF4A overexpression in the HNF4A-negative IHH and knockdown on primary hepatocytes. The results showed that morphology, hepatic function, and differentiation of IHH were notably enhanced by HNF4A overexpression. Surprisingly, HNF4A overexpression was associated with down-regulation of c-myc and chromosome instability in primary human hepatocytes treated with HNF4A shRNA. Based on a previous study [7,38] and the present study, we propose that HNF4A can improve hepatic differentiation and function of IHH. Wang et al. [14] reported that c-myc expression was up-regulated in hTERT-immortalized human mammary epithelial cells between 107 and 135 PD. They concluded that extension of lifespan by telomerase reconstitution might select c-myc overexpressing cells. Wege et al. [8] found that telomerase reconstitution did not result in c-myc up-regulation after 160 PD and indicated that telomerase reconstitution did not induce a transformed phenotype and was likely to be genoprotective throughout the expanded proliferative lifespan of immortalized cells. Wang et al. [14] consider c-myc as an important tumorigenicity marker. Nevertheless, it can be inferred from the present study that HNF4A likely regulates the expression of c-myc, although the mechanism remains elusive. This implies that the expression of HNF4A in hepatocytes is inversely related to the activation of the c-myc gene (Fig. 6C). When HNF4A is negative, the expression of c-Myc is upregulated, and phenotypic abnormalities and chromosomal instability are then present (Fig. 6D), Eventually, the hepatocytes showed 10

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(caption on next page)

Wnt/β-catenin signaling.

Acknowledgements

Conflict of interests

This project was supported by the National Natural Science Foundation of China (No. 81570561), Shanghai Pujiang Program (17PJD023) and Clinical Research Plan of SHDC (16CR3106B).

The authors declare no conflict of interests. 11

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Fig. 8. Transplantation of encapsulated HNF4A2-negative IHH overexpressing HNF4A into rat models of acute liver failure (ALF). (A) Morphology of encapsulated IHH. Empty microencapsulation (magnification × 200). (B) IHH were encapsulated in alginate-poly-L-lysine-alginate microcapsules on day 0 (magnification × 200). The rats were randomly divided into three groups. In the empty capsules group (n = 20), 2 ml of empty capsules were injected into the peritoneal cavity. In the encapsulated IHH group (n = 20), 5 × 107 encapsulated IHH were injected into the peritoneal cavity. In the encapsulated IHH with HNF4α overexpression group (n = 20), 5 × 107 encapsulated IHH overexpressing HNF4A were injected into the peritoneal cavity. Twenty-four hours after transplantation, the empty capsule group experienced a rapid increase in blood ALT (C), AST (D), and total bilirubin (TB) (E) levels. In contrast, these parameters among rats with cell transplantation (both IHH groups) were markedly decreased at the same time points, especially in the HNF4 overexpressing IHH group. *P < 0.05, **P < 0.01 vs. empty capsules group; #P < 0.05 vs. encapsulated IHH group. (F) Comparative survival curves 8 days post induction of ALF and IHH transplantation. Peak of death was on the third day in the empty capsule group, the 5th day in the normal IHH group, and the 6th day in the HNF4 overexpressing IHH group, respectively. **P < 0.01 encapsulated IHH group vs. empty capsules group; *P < 0.05 encapsulated IHH with HNF4α overexpression group vs. encapsulated IHH group. (G) Liver specimens obtained from the rats 24 h after of D-Gal administration. Massive hemorrhage and extensive hepatocytes necrosis were observed in the portal areas (magnification × 200). (H) Liver samples taken from the normal IHH group 24 h after cell transplantation. Hemorrhage and hepatocytes necrosis were improved after transplantation of encapsulated IHH (magnification × 200). (I) Liver specimens obtained from the HNF4 overexpressing IHH group 24 h after cell transplantation. Only mild hemorrhage and hepatocytes necrosis were observed in the portal areas after transplantation of encapsulated IHH overexpressing HNF4α (magnification × 200). Asterisk: massive hemorrhage; arrowhead: extensive hepatocytes necrosis.

[22] Y. Inoue, A.M. Yu, S.H. Yim, X. Ma, K.W. Krausz, J. Inoue, et al., Regulation of bile acid biosynthesis by hepatocyte nuclear factor 4alpha, J. Lipid Res. 47 (2006) 215–227. [23] J.A. Ladias, M. Hadzopoulou-Cladaras, D. Kardassis, P. Cardot, J. Cheng, V. Zannis, et al., Transcriptional regulation of human apolipoprotein genes ApoB, ApoCIII, and ApoAII by members of the steroid hormone receptor superfamily HNF-4, ARP-1, EAR-2, and EAR-3, J. Biol. Chem. 267 (1992) 15849–15860. [24] F.M. Sladek, M.D. Ruse Jr., L. Nepomuceno, S.M. Huang, M.R. Stallcup, Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1, Mol. Cell Biol. 19 (1999) 6509–6522. [25] C.K. Glass, M.G. Rosenfeld, The coregulator exchange in transcriptional functions of nuclear receptors, Genes Dev. 14 (2000) 121–141. [26] H. Hang, X. Shi, G. Gu, Y. Wu, Y. Ding, A simple isolation and cryopreservation method for adult human hepatocytes, Int. J. Artif. Organs 32 (2009) 720–727. [27] H. Hang, X. Shi, G. Gu, Y. Wu, J. Gu, Y. Ding, In vitro analysis of cryopreserved alginate-poly-L-lysine-alginate-microencapsulated human hepatocytes, Liver Int. 30 (2010) 611–622. [28] F.W. Vondran, E. Katenz, R. Schwartlander, M.H. Morgul, N. Raschzok, X. Gong, et al., Isolation of primary human hepatocytes after partial hepatectomy: criteria for identification of the most promising liver specimen, Artif. Organs 32 (2008) 205–213. [29] K. Dorko, P.D. Freeswick, F. Bartoli, L. Cicalese, B.A. Bardsley, A. Tzakis, et al., A new technique for isolating and culturing human hepatocytes from whole or split livers not used for transplantation, Cell Transplant. 3 (1994) 387–395. [30] V.M. Tesmer, L.P. Ford, S.E. Holt, B.C. Frank, X. Yi, D.L. Aisner, et al., Two inactive fragments of the integral RNA cooperate to assemble active telomerase with the human protein catalytic subunit (hTERT) in vitro, Mol. Cell Biol. 19 (1999) 6207–6216. [31] S. Cereghini, Liver-enriched transcription factors and hepatocyte differentiation, FASEB J. 10 (1996) 267–282. [32] Y.S. Fan, P. Li, Cytogenetic studies of four human lung adenocarcinoma cell lines, Cancer Genet. Cytogenet. 26 (1987) 317–325. [33] X.L. Shi, Y. Zhang, J.Y. Gu, Y.T. Ding, Coencapsulation of hepatocytes with bone marrow mesenchymal stem cells improves hepatocyte-specific functions, Transplantation 88 (2009) 1178–1185. [34] B.C. Godthelp, P.P. van Buul, N.G. Jaspers, E. Elghalbzouri-Maghrani, A. van DuijnGoedhart, F. Arwert, et al., Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2, Mutat. Res. 601 (2006) 191–201. [35] E. Krieghoff, J. Behrens, B. Mayr, Nucleo-cytoplasmic distribution of beta-catenin is regulated by retention, J. Cell Sci. 119 (2006) 1453–1463. [36] L. Xu, L. Hui, S. Wang, J. Gong, Y. Jin, Y. Wang, et al., Expression profiling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma, Cancer Res. 61 (2001) 3176–3181. [37] P. Flodby, D.Z. Liao, A. Blanck, K.G. Xanthopoulos, I.P. Hallstrom, Expression of the liver-enriched transcription factors C/EBP alpha, C/EBP beta, HNF-1, and HNF-4 in preneoplastic nodules and hepatocellular carcinoma in rat liver, Mol. Carcinog. 12 (1995) 103–109. [38] N.L. Lazarevich, O.A. Cheremnova, E.V. Varga, D.A. Ovchinnikov, E.I. Kudrjavtseva, O.V. Morozova, et al., Progression of HCC in mice is associated with a downregulation in the expression of hepatocyte nuclear factors, Hepatology 39 (2004) 1038–1047. [39] J. Li, L.J. Li, H.C. Cao, G.P. Sheng, H.Y. Yu, W. Xu, et al., Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy, ASAIO J. 51 (2005) 262–268. [40] P. Salmon, J. Oberholzer, T. Occhiodoro, P. Morel, J. Lou, D. Trono, Reversible immortalization of human primary cells by lentivector-mediated transfer of specific genes, Mol. Ther. 2 (2000) 404–414. [41] T. Watanabe, N. Shibata, K.A. Westerman, T. Okitsu, J.E. Allain, M. Sakaguchi, et al., Establishment of immortalized human hepatic stellate scavenger cells to develop bioartificial livers, Transplantation 75 (2003) 1873–1880. [42] C.A. Lazaro, E.J. Croager, C. Mitchell, J.S. Campbell, C. Yu, J. Foraker, et al., Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes, Hepatology 38 (2003) 1095–1106. [43] R. Iacob, U. Rudrich, M. Rothe, S. Kirsch, B. Maasoumy, N. Narain, et al., Induction of a mature hepatocyte phenotype in adult liver derived progenitor cells by ectopic expression of transcription factors, Stem Cell Res. 6 (2011) 251–261. [44] M. Peifer, P. Polakis, Wnt signaling in oncogenesis and embryogenesis–a look outside the nucleus, Science 287 (2000) 1606–1609.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2017.08.020. References [1] M.P. Manns, Liver cirrhosis, transplantation and organ shortage, Dtsch. Arztebl. Int. 110 (2013) 83–84. [2] R.D. Hughes, R.R. Mitry, A. Dhawan, Current status of hepatocyte transplantation, Transplantation 93 (2012) 342–347. [3] N. Arkadopoulos, S.C. Chen, T.M. Khalili, O. Detry, W.R. Hewitt, H. Lilja, et al., Transplantation of hepatocytes for prevention of intracranial hypertension in pigs with ischemic liver failure, Cell Transplant. 7 (1998) 357–363. [4] Y. Ilan, Towards a bank of cryopreserved hepatocytes: which cell to freeze? J. Hepatol. 37 (2002) 145–146. [5] H.B. Stockmann, I.J. JN, Prospects for the temporary treatment of acute liver failure, Eur. J. Gastroenterol. Hepatol. 14 (2002) 195–203. [6] N. Kobayashi, T. Fujiwara, K.A. Westerman, Y. Inoue, M. Sakaguchi, H. Noguchi, et al., Prevention of acute liver failure in rats with reversibly immortalized human hepatocytes, Science 287 (2000) 1258–1262. [7] T. Totsugawa, C. Yong, J.D. Rivas-Carrillo, A. Soto-Gutierrez, N. Navarro-Alvarez, H. Noguchi, et al., Survival of liver failure pigs by transplantation of reversibly immortalized human hepatocytes with Tamoxifen-mediated self-recombination, J. Hepatol. 47 (2007) 74–82. [8] H. Wege, H.T. Le, M.S. Chui, L. Liu, J. Wu, R. Giri, et al., Telomerase reconstitution immortalizes human fetal hepatocytes without disrupting their differentiation potential, Gastroenterology 124 (2003) 432–444. [9] Y. Tsuruga, T. Kiyono, M. Matsushita, T. Takahashi, H. Kasai, S. Matsumoto, et al., Establishment of immortalized human hepatocytes by introduction of HPV16 E6/E7 and hTERT as cell sources for liver cell-based therapy, Cell Transplant. 17 (2008) 1083–1094. [10] M. Maruyama, N. Kobayashi, K.A. Westerman, M. Sakaguchi, J.E. Allain, T. Totsugawa, et al., Establishment of a highly differentiated immortalized human cholangiocyte cell line with SV40T and hTERT, Transplantation 77 (2004) 446–451. [11] E. Ramboer, T. Vanhaecke, V. Rogiers, M. Vinken, Immortalized human hepatic cell lines for in vitro testing and research purposes, Methods Mol. Biol. 1250 (2015) 53–76. [12] C. Fauth, M.J. O'Hare, G. Lederer, P.S. Jat, M.R. Speicher, Order of genetic events is critical determinant of aberrations in chromosome count and structure, Genes Chromosomes Cancer 40 (2004) 298–306. [13] L.F. Meisner, S.Q. Wu, B.J. Christian, C.A. Reznikoff, Cytogenetic instability with balanced chromosome changes in an SV40 transformed human uroepithelial cell line, Cancer Res. 48 (1988) 3215–3220. [14] J. Wang, G.J. Hannon, D.H. Beach, Risky immortalization by telomerase, Nature 405 (2000) 755–756. [15] T.H. Nguyen, G. Mai, P. Villiger, J. Oberholzer, P. Salmon, P. Morel, et al., Treatment of acetaminophen-induced acute liver failure in the mouse with conditionally immortalized human hepatocytes, J. Hepatol. 43 (2005) 1031–1037. [16] J.A. Bonzo, C.H. Ferry, T. Matsubara, J.H. Kim, F.J. Gonzalez, Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4alpha in adult mice, J. Biol. Chem. 287 (2012) 7345–7356. [17] C. Cicchini, L. Amicone, T. Alonzi, A. Marchetti, C. Mancone, M. Tripodi, Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte, Liver Int. 35 (2015) 302–310. [18] J. Li, G. Ning, S.A. Duncan, Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha, Genes Dev. 14 (2000) 464–474. [19] F. Parviz, C. Matullo, W.D. Garrison, L. Savatski, J.W. Adamson, G. Ning, et al., Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis, Nat. Genet. 34 (2003) 292–296. [20] D.T. Odom, N. Zizlsperger, D.B. Gordon, G.W. Bell, N.J. Rinaldi, H.L. Murray, et al., Control of pancreas and liver gene expression by HNF transcription factors, Science 303 (2004) 1378–1381. [21] Y. Inoue, L.L. Peters, S.H. Yim, J. Inoue, F.J. Gonzalez, Role of hepatocyte nuclear factor 4alpha in control of blood coagulation factor gene expression, J. Mol. Med. 84 (2006) 334–344.

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Experimental Cell Research xxx (xxxx) xxx–xxx

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[48] P. Polakis, Wnt signaling in cancer, Cold Spring Harb. Perspect. Biol. (2012) 4. [49] B.F. Ning, J. Ding, C. Yin, W. Zhong, K. Wu, X. Zeng, et al., Hepatocyte nuclear factor 4 alpha suppresses the development of hepatocellular carcinoma, Cancer Res. 70 (2010) 7640–7651. [50] C. Yin, Y. Lin, X. Zhang, Y.X. Chen, X. Zeng, H.Y. Yue, et al., Differentiation therapy of hepatocellular carcinoma in mice with recombinant adenovirus carrying hepatocyte nuclear factor-4alpha gene, Hepatology 48 (2008) 1528–1539.

[45] P.J. Morin, beta-catenin signaling and cancer, Bioessays 21 (1999) 1021–1030. [46] S.Z. Hussain, T. Sneddon, X. Tan, A. Micsenyi, G.K. Michalopoulos, S.P. Monga, Wnt impacts growth and differentiation in ex vivo liver development, Exp. Cell Res. 292 (2004) 157–169. [47] S.P. Monga, A. Micsenyi, M. Germinaro, U. Apte, A. Bell, beta-Catenin regulation during matrigel-induced rat hepatocyte differentiation, Cell Tissue Res. 323 (2006) 71–79.

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