Infection of a human hepatoma cell line by hepatitis B virus

Among numerous established human hepatoma cell lines, none has been shown susceptible to hepatitis B virus (HBV) infection. We describe here a cell line, called HepaRG, which exhibits hepatocyte-like morphology, expresses specific hepatocyte functions, and supports HBV infection as well as primary cultures of normal human hepatocytes. Differentiation and infectability are maintained only when these cells are cultured in the presence of corticoids and dimethyl sulfoxide. The specificity of this HBV infection model was ascertained by both the neutralization capacity of HBV-envelope protein-specific antibodies and the competition with an envelope-derived peptide. HepaRG cells therefore represent a tool for deciphering the mechanism of HBV entry. Moreover, their close resemblance to normal human hepatocytes makes them suitable for many applications including drug metabolism studies.     

The HepaRG cell line: a valuable in vitro tool for hepatitis virus infection studies

Hepatitis virus infections, mainly hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, constitute a major problem for public health since they have a worldwide distribution and because they are associated with hepatocellular carcinoma and death. Current anti-HBV vaccines seem to be effective in the majority of people. However, an important issue waiting to be tackled nowadays is how to cure patients with chronic hepatitis B. Moreover, no vaccine is available today for the prevention of HCV infection. Therefore, the use of adequate in vitro infection systems is a prerequisite for the molecular understanding of the infection events of these viruses, which could result in the development of novel powerful therapeutics. In this respect, the HepaRG cell line exhibits a hepatocyte-like morphology and displays drug metabolism capacity similar to that of primary hepatocytes. HepaRG cells have yet been proven to be a useful tool in the study of viral infections, particularly for deciphering the mechanism of HBV entry into hepatocytes.     

Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.     

Primary human hepatocytes--a valuable tool for investigation of apoptosis and hepatitis B virus infection

BACKGROUND/AIMS: Apoptosis is a key event in the pathophysiology of many liver diseases. Primary human hepatocytes (PHH) provide a useful model to study physiological and pathophysiological processes in the liver. Our aim was to optimize PHH cultures to allow studies on induction of apoptosis and of hepatitis B virus (HBV) infection. METHODS: PHH were isolated from human liver tissue by two-step collagenase perfusion. PHH and hepatoma cells were treated with different apoptosis-inducing agents in parallel. PHH cultures were infected with wild type HBV and transduced with HBV genomes using adenoviral vectors. RESULTS: PHH were successfully isolated from 40 different tissue samples with high viability and purity. Perfusion time and seeding density turned out to be critical parameters for optimal cell yield and culture conditions, respectively. Serum addition to the medium reduced viability of PHH. PHH allowed reproducible studies of CD95-dependent and -independent apoptosis. Sensitivity towards CD95-mediated apoptosis was markedly higher than in hepatoma cells. PHH could efficiently be infected with HBV, but infection did neither induce apoptosis nor prevent CD95-induced cell death. CONCLUSIONS: Our data show that PHH provide an excellent tool for the investigation of apoptosis induced by agents like death receptor-ligands and hepatotropic viruses.     

Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans

Contrary to many other viruses, the initial steps of the hepatitis B virus (HBV) infection, including attachment to hepatocytes, specific receptor interactions, and membrane fusion, are unsolved. Using HepaRG cells as an in vitro cell culture system, we here report that HBV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cell-surface-associated heparan sulfate proteoglycans. Binding to GAGs requires the integrity of the pre-S domain as a part of the large (L-) viral envelope protein. HBV infection was abrogated by incubation of virions with heparin, but not the structurally related GAGs chondroitin sulfate A, B, and C. Infection was also abolished by suramin, a known inhibitor of duck hepatitis B virus infection or highly sulfated dextran sulfate. Polycationic substances such as poly-L-lysine, polybrene, and protamine also prevented infection, however, by addressing cellular components. Enzymatic removal of defined acidic carbohydrate structures from the cell surface using heparinase I/III or the obstruction of GAG synthesis by sodium chlorate inhibited HBV infection of HepaRG cells and, moreover, led to a reduction of HBV cell surface binding sites. The biochemical analysis showed selective binding of L-protein-enriched viral particles (virions or filaments) to heparin. GAG-dependent binding of HBV was improved by polyethylene glycol, a substance that specifically enhances HBV infection. Conclusion: HBV infection requires the initial attachment to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors. This interaction initializes the multistep entry process of HBV and cannot be bypassed by alternative routes.     

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes

HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors.     

Characterization of a hepatitis B and hepatitis delta virus receptor binding site

Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion- and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) a myristoylated HBVpreS/2-48-GST fusion protein efficiently interferes with HBV infection of HepaRG cells; (2) deletions and point mutations in the highly conserved preS1 sequence between amino acids 11 through 21 result in the loss of infection inhibition activity; (3) hepatitis B viruses carrying single amino acid exchanges within this region lose infectivity; and (4) HDV infection of HepaRG cells can be inhibited by myristoylated HBVpreS peptides with the same specificity. In conclusion, HBV and HDV use at least one common step to enter hepatocytes and require a highly conserved preS1-sequence within the L-protein. This step is exceptionally sensitive toward inactivation by acylated HBVpreS1 peptides, which therefore represent a novel group of entry inhibitors that could be used for the treatment of hepatitis B and D.     

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.     

Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

AIMTo investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODSThe human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTSA stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSIONHBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.     

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection

The Na⁺/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.     

HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in Western countries. In the last four decades much progress has been made in our understanding of APAP-induced liver injury through rodent studies. However, some differences exist in the time course of injury between rodents and humans. To study the mechanism of APAP hepatotoxicity in humans, a human-relevant in vitro system is needed. Here we present evidence that the cell line HepaRG is a useful human model for the study of APAP-induced liver injury. Exposure of HepaRG cells to APAP at several concentrations resulted in glutathione depletion, APAP-protein adduct formation, mitochondrial oxidant stress and peroxynitrite formation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase (LDH) release. Importantly, the time course of LDH release resembled the increase in plasma aminotransferase activity seen in humans following APAP overdose. Based on propidium iodide uptake and cell morphology, the majority of the injury occurred within clusters of hepatocyte-like cells. The progression of injury in these cells involved mitochondrial reactive oxygen and reactive nitrogen formation. APAP did not increase caspase activity above untreated control values and a pancaspase inhibitor did not protect against APAP-induced cell injury. CONCLUSION: These data suggest that key mechanistic features of APAP-induced cell death are the same in human HepaRG cells, rodent in vivo models, and primary cultured mouse hepatocytes. Thus, HepaRG cells are a useful model to study mechanisms of APAP hepatotoxicity in humans.     

High expression of hepatitis B virus based vector with reporter gene in hepatitis B virus infection system

AIM: To construct a hepatitis B virus (HBV)-based vector with a reporter gene and to establish an HBV infection system to evaluate the availability of the vector. METHODS: The HBV-based vectors with green fluorescence protein (GFP) were packaged into the liver of immunodeficient mice through transfer and helper plasmid using hydrodynamic technology. Wild type HBV (wt HBV) was provided by plasmid MC2009. Primary human hepatocytes (PHH) were isolated and infected with recombinant HBV (rHBV) or wt HBV. GFP expression was monitored by confocal and flow cytometry. HBV DNA and HBV surface antigen (HBSAg) were analyzed by PCR and ELISA. RESULTS: 3 × 107 wt HBV copies/mL and 5 × 106 rHBV copies/mL were collected from mice serum. In the wt HBV infected group, HBV progeny was 2 × 107 copies/mL and HBSAg was 770 ng/mL. In the rHBV infected group, GFP fluorescence was detected on d 3 post-infection and over 85% of the parenchymal cells expressed green fluorescence on d 12 post-infection. Compared with wt HBV in the PHH infection system, no rHBV DNA or HBSAg were detected in PHH culture media. CONCLUSION: An effective HBV based vector was developed, which proved to be a useful HBV infection system. This vector and infection system can be applied to develop a therapeutic vector and study the HBV life cycle and viral pathogenesis.     

Origin and characterization of a human bipotent liver progenitor cell line

BACKGROUND & AIMS: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. METHODS: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. RESULTS: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. CONCLUSIONS: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.     

HBV culture and infectious systems

While an effective vaccine against hepatitis B virus (HBV) has long been available, chronic HBV infection remains a severe global public health concern. Current treatment options have limited effectiveness, and long-term therapy is required to suppress HBV replication; however, complete elimination of the virus is rare. The lack of suitable animal models and infection systems has hindered efforts to unravel the HBV life cycle, particularly the early events in HBV entry, which appear to be highly species- and tissue-specific. Human primary hepatocytes remain the gold standard for HBV replication studies but are limited by availability and variability. While the HepaRG cell line is permissive for HBV replication, other hepatoma cell lines such as HepG2 do not support HBV replication. The recent discovery of sodium taurocholate transporting peptide (NTCP) as a primary receptor for HBV binding has led to the development of replication-competent cell lines such as HepG2–NTCP. Human hepatocytes grown in chimeric mice have provided another approach that allows primary human hepatocytes to be used while overcoming many of their limitations. Although the difficulty in developing HBV infection systems has hindered development of effective treatments, the variability and limited replication efficiency among cell lines point to additional liver-specific factors involved in HBV infection. It is hoped that HBV infection studies will lead to novel drug targets and therapeutic options for the treatment of chronic HBV infection.     

Viral and cellular determinants involved in hepadnaviral entry

Hepadnaviridae is a family of hepatotropic DNA viruses that is divided into the genera orthohepadnavirus of mammals and avihepadnavirus of birds. All members of this family can cause acute and chronic hepatic infection, which in the case of human hepatitis B virus (HBV) constitutes a major global health problem. Although our knowledge about the molecular biology of these highly liver-specific viruses has profoundly increased in the last two decades, the mechanisms of attachment and productive entrance into the differentiated host hepatocytes are still enigmatic. The difficulties in studying hepadnaviral entry were primarily caused by the lack of easily accessible in vitro infection systems. Thus, for more than twenty years, differentiated primary hepatocytes from the respective species were the only in vitro models for both orthohepadnaviruses (e.g. HBV) and avihepadnaviruses (e.g. duck hepatitis B virus [DHBV]). Two important discoveries have been made recently regarding HBV: (1) primary hepatocytes from tree-shrews; i.e., Tupaia belangeri, can be substituted for primary human hepatocytes, and (2) a human hepatoma cell line (HepaRG) was established that gains susceptibility for HBV infection upon induction of differentiation in vitro. A number of potential HBV receptor candidates have been described in the past, but none of them have been confirmed to function as a receptor. For DHBV and probably all other avian hepadnaviruses, carboxypeptidase D (CPD) has been shown to be indispensable for infection, although the exact role of this molecule is still under debate. While still restricted to the use of primary duck hepatocytes (PDH), investigations performed with DHBV provided important general concepts on the first steps of hepadnaviral infection. However, with emerging data obtained from the new HBV infection systems, the hope that DHBV utilizes the same mechanism as HBV only partially held true. Nevertheless, both HBV and DHBV in vitro infection systems will help to: (1) functionally dissect the hepadnaviral entry pathways, (2) perform reverse genetics (e.g. test the fitness of escape mutants), (3) titrate and map neutralizing antibodies, (4) improve current vaccines to combat acute and chronic infections of hepatitis B, and (5) develop entry inhibitors for future clinical applications.     

Definition of an HBsAg to DNA international unit conversion factor by enrichment of circulating hepatitis B virus forms

Hepatitis B (HBV) virus infection is characterized by the overproduction of subviral particles (SVP) over infectious Dane particles (VP). Precise regulation of the ratio between these forms is unknown, but its fluctuation may have a clinical impact. An enrichment method was applied to assess the SVP/VP ratio in chronically infected patients (CHB) and to compare the sensitivity of HBs antigen (HBsAg) and DNA detection methods. Plasmas from 9 genotype A–D CHB patients were fractionated on Nycodenz^® gradients, and both HBV DNA and HBsAg were quantified in each collected fraction using standardized techniques expressed in IU/mL. Infection of primary human hepatocytes (PHHs) was performed with crude or fractionated plasma. Independently of the genotype, all plasmas showed a similar rate‐zonal separation profile characterized by a bottom DNA‐enriched peak surmounted by HBsAg‐enriched fractions. Inoculation of PHH with plasma‐derived VP‐enriched fractions led to long‐lasting production of virus in cell supernatants with a SVP/VP ratio similar to that observed in patient plasmas. In the VP fraction, one IU of HBsAg corresponded to approximately 5 million IU of HBV DNA. Rate‐zonal gradient separation directly applied on patient plasma allows a better insight into the distribution of VP in HBeAg‐positive CHB carriers. This study highlights the sensitivity difference of the techniques classically used to monitor HBV infection and indicates that VP‐associated HBsAg contributes modestly to the overall amount of total circulating HBsAg in CHB. Such a fractionation approach should help to understand the fine regulation of HBsAg production over replication at different stages of CHB.     

Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes

Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.Drug-induced liver injury (DILI) is a leading cause of the withdrawal of drugs from the market. Human induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) are expected to be useful for the prediction of DILI in the early phase of drug development. Many groups, including our own, have reported that the human iPS-HLCs have the ability to metabolize drugs, and thus these cells could be used to detect the cytotoxicity of drugs that are known to cause DILI (1, 2). However, to accurately predict DILI, it will be necessary to establish a panel of human iPS-HLCs that better represents the genetic variation of the human population because there are large interindividual differences in the drug metabolism capacity and drug responsiveness of hepatocytes (3). However, it remains unclear whether the drug metabolism capacity and drug responsiveness of human iPS-HLCs could reflect those of donor parental primary human hepatocytes (PHHs). To address this issue, we generated the HLCs differentiated from human iPSCs which had been established from PHHs (PHH-iPS-HLCs). Then, we compared the drug metabolism capacity and drug responsiveness of PHH-iPS-HLCs with those of their parental PHHs, which are genetically identical to the PHH-iPS-HLCs.Interindividual differences of cytochrome P450 (CYP) metabolism capacity are closely related to genetic polymorphisms, especially single nucleotide polymorphisms (SNPs), in CYP genes (4). Among the various CYPs expressed in the liver, CYP2D6 is responsible for the metabolism of approximately a quarter of commercially used drugs and has the largest phenotypic variability, largely due to SNPs (5). It is known that certain alleles result in the poor metabolizer phenotype due to a decrease of CYP2D6 metabolism. Therefore, the appropriate dosage for drugs that are metabolized by CYP2D6, such as tamoxifen, varies widely among individuals (6). Indeed, in the 1980s, polymorphism in CYP2D6 appears to have contributed to the withdrawal of CYP2D6-metabolized drugs such as perhexiline from the market in many countries (7). If we could establish a panel of HLCs that better represents the diversity of genetic polymorphisms in the human population, it might be possible to determine the appropriate dosage of a drug for a particular individual. However, it is not known whether the drug metabolism capacity and drug responsiveness of HLCs reflect the genetic diversity, including SNPs, in CYP genes. Therefore, in this study we generated HLCs from several PHHs that have various SNPs on CYP2D6 and then compared the CYP2D6 metabolism capacity and responses to CYP2D6-metabolized drugs between the PHH-iPS-HLCs and parental PHHs.To this end, PHHs were reprogrammed into human iPSCs and then differentiated into the HLCs. To examine whether the HLCs could reproduce the characteristics of donor PHHs, we first compared the CYP metabolism capacity and response to a hepatotoxic drug between PHHs and genetically identical PHH-iPS-HLCs (12 donors were used in this study). Next, analyses of hepatic functions, including comparisons of the gene expression of liver-specific genes and CYPs, were performed to examine whether the hepatic characteristics of PHHs were reproduced in the HLCs. To the best of our knowledge, this is the first study to compare the functions between iPSC-derived cells from various donors and their parental cells with identical genetic backgrounds. Finally, we examined whether the PHH-iPS-HLCs exhibited a capacity for drug metabolism and drug responsiveness that reflect the genetic diversity such as SNPs on CYP genes.     

Foxp3+ regulatory T cells protect the liver from immune damage and compromise virus control during acute experimental hepatitis B virus infection in mice

The strength of antiviral T cell responses correlates with clearance of hepatitis B virus (HBV) infection, but the immunological mechanisms mitigating or suppressing HBV-specific T cells are still poorly understood. In this study, we examined the role of CD4(+) Foxp3(+) regulatory T cells (Tregs) in a mouse model of acute HBV infection. We initiated HBV infection via an adenoviral vector transferring a 1.3-fold overlength HBV genome (AdHBV) into transgenic DEREG mice, where Tregs can be transiently but selectively depleted by injection of diphtheria toxin. The effect of Treg depletion on the outcome of HBV infection was characterized by detailed virological, immunological, and histopathological analysis. Numbers of Tregs increase in the liver rapidly after initiation of HBV replication. Initial depletion of Tregs revealed their complex regulatory function during acute infection. Tregs mitigated immunomediated liver damage by down-regulating the antiviral activity of effector T cells by limiting cytokine production and cytotoxicity, but did not influence development of HBV-specific CD8 T cells or development of memory T cells. Furthermore, Tregs controlled the recruitment of innate immune cells such as macrophages and dendritic cells to the infected liver. As a consequence, Tregs significantly delayed clearance of HBV from blood and infected hepatocytes. Conclusion: Tregs limit immunomediated liver damage early after an acute infection of the liver, thereby contributing to conservation of tissue integrity and organ function at the cost of prolonging virus clearance. (HEPATOLOGY 2012;56:873-883).