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Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4 , leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Background: MicroRNAs (miRNAs) have gained much attention as cellular factors regulating hepatitis C virus (HCV) infection. miR-27b has been shown to regulate HCV infection in the hepatocytes via various mechanisms that have not been fully elucidated. In this study, therefore, we examined the mechanisms of miR-27b-mediated regulation of HCV infection., Methods: In silico screening analysis, transfection with miR-27b mimic, and a cell-based reporter assay were performed to identify miR-27b target genes. Cell cultured-derived HCV (HCVcc) was added to Huh7.5.1 cells knocked down for aquaporin (AQP) 11 (AQP11) and overexpressing AQP11. HCV replication levels were evaluated by real-time RT-PCR analysis of HCVcc genome., Results: Infection of Huh7.5.1 cells with HCVcc resulted in significant elevation in miR-27b expression levels. In silico analysis revealed that AQP11, which is an AQP family member and is mainly localized in the endoplasmic reticulum (ER), was a candidate for a target gene of miR-27b. Transfection of a miR-27b mimic significantly reduced AQP11 expression, but a cell-based reporter assay demonstrated that miR-27b did not suppress the expression of a reporter gene containing the 3'-untranslated region of the AQP11 gene, suggesting that miR-27b indirectly suppressed AQP11 expression. AQP11 expression levels were significantly reduced by infection with HCVcc in Huh7.5.1 cells. Knockdown and over-expression of AQP11 significantly reduced and increased HCVcc genome levels in the cells following infection, respectively, however, AQP11 knockdown did not show significant effects on the HCVcc titers in the culture supernatants., Conclusions: These results indicated that HCV infection induced a miR-27b-mediated reduction in AQP11 expression, leading to a modest reduction in HCV genome levels in the cells, not HCV titers in the culture supernatants.

Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.

Hepatocyte-like cells differentiated from human iPS cells are expected to be utilized in pharmaceutical research and regenerative medicine. Recently, various culture methods for human iPS cell maintenance have been developed. However, it is not well known whether human iPS cell maintenance method affects hepatic differentiation potency. In this study, we cultured human iPS cells using four maintenance methods: ReproStem medium with feeder cells (mouse embryonic fibroblasts), AK02N medium with iMatrix-511 (E8 fragments of laminin511), Essential 8 medium with Vitronectin N (N-terminal domain of vitronectin), TeSR-E8 medium with Vitronectin XF (xeno-free vitronectin). Then, these human iPS cells were differentiated into the hepatocyte-like cells. Interestingly, the gene expression levels of definitive endoderm markers in the definitive endoderm cells generated from human iPS cells cultured with ReproStem or TeSR-E8 medium were higher than those in other groups. The gene expression level of foregut marker, HHEX, in the definitive endoderm cells generated from human iPS cells cultured with ReproStem medium was higher than that in other groups. Consistently, the expression levels of hepatocyte markers, albumin and urea secretion capacity, and CYP3A4 activity in the hepatocyte-like cells generated from human iPS cells cultured with ReproStem medium were higher than those in the other groups. Our data indicated that the most suitable human iPS cell maintenance method for efficient hepatic differentiation was the on-feeder method which uses mouse embryonic fibroblasts, but not feeder-free methods. In conclusion, human iPS cell maintenance method largely affects hepatic differentiation potency., (Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Human iPS cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research. However, the purity of high-functioning hepatocyte-like cells is not high enough. In particular, the purity of cytochrome P450 3A4 (CYP3A4), which is a representative hepatic drug-metabolizing enzyme, positive cells is still quite low (approximately 20%). To address this problem, we established the CYP3A4-NeoR-EGFP transgenic reporter human iPS cell line (CYP3A4-NeoR-EGFP iPS cells) by using genome editing technology. The CYP3A4-NeoR-EGFP iPS cells were differentiated into hepatocyte-like cells, and then the hepatocyte-like cells were treated with neomycin to concentrate the hepatocyte-like cells which strongly express CYP3A4. After the neomycin treatment, the percentage of CYP3A4-positive cells was higher than 80%. The gene expression levels of various drug-metabolizing enzymes, transporters, and hepatic transcription factors were significantly enhanced by neomycin treatment. In addition, the CYP1A2, 2C19, 2D6, and 3A4 activities and biliary excretion capacities were significantly increased by neomycin treatment. We also confirmed that the detection sensitivity of drug-inducing hepatotoxicity was enhanced by neomycin treatment. We succeeded in obtaining human iPS cell-derived hepatocyte-like cells that highly express CYP3A4 at high purity. We believe that our high-purity and high-functioning hepatocyte-like cells could be used to evaluate the risk of drug candidates., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) are expected to be utilized in pharmaceutical research, including drug screening. However, the hepatocyte functions of the HLCs are still lower than those of human hepatocytes. Therefore, we attempted to improve the hepatocyte differentiation method by modulating the DNA epigenetic status. We first examined the expression profiles of the maintenance DNA methyltransferase (DNMT) 1 and the de novo DNMTs DNMT3A and DNMT3B, all of which are essential for mammalian development. Among these DNMTs, the expression levels of DNMT3B were significantly decreased during the hepatoblast differentiation. To accelerate the hepatoblast differentiation, a DNMT3B-selective inhibitor, nanaomycin A, was treated during the hepatoblast differentiation. The gene expression levels of hepatoblast markers (such as alpha-fetoprotein and hepatocyte nuclear factor 4 alpha) were increased by the nanaomycin A treatment. On the other hand, the gene expression levels of hepatoblast markers were decreased by DNMT3B overexpression. These results suggest that it might be possible to promote the hepatoblast differentiation by DNMT3B inhibition using nanaomycin A. Importantly, we also confirmed that the hepatocyte differentiation potency of nanaomycin A-treated hepatoblast-like cells was higher than that of dimethyl sulfoxide-treated hepatoblast-like cells. Our findings should assist in the future generation of functional HLCs for pharmaceutical research.

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells are expected to be utilized in drug screening and regenerative medicine. However, hepatocyte-like cells have not been fully used in such applications because it is difficult to produce such cells on a large scale. In this study, we tried to establish a method to mass produce hepatocyte-like cells using a three-dimensional (3D) cell culture bioreactor called the Rotary Cell Culture System (RCCS). RCCS enabled us to obtain homogenous hepatocyte-like cells on a billion scale (>10 9  cells). The gene expression levels of some hepatocyte markers (alpha-1 antitrypsin, cytochrome (CYP) 1A2, CYP2D6, and hepatocyte nuclear factor 4alpha) were higher in 3D-cultured hepatocyte-like cells than in 2D-cultured hepatocyte-like cells. This result suggests that RCCS could provide more suitable conditions for hepatocyte maturation than the conventional 2D cell culture conditions. In addition, more than 90% of hepatocyte-like cells were positive for albumin and could uptake low-density lipoprotein in the culture medium. We succeeded in the large-scale production of homogenous and functional hepatocyte-like cells from human iPS cells. This technology will be useful in drug screening and regenerative medicine, which require enormous numbers of hepatocyte-like cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recently, it has been reported that human hepatocyte-like cells can be generated from fibroblasts by direct reprogramming technology. However, the conversion efficiency of human induced hepatocyte-like cells (hiHeps) is not high enough. In addition, comparative analysis with the existing models of hepatocytes, such as human iPS cell-derived hepatocyte-like cells and primary human hepatocytes, has not been sufficiently carried out. In this study, we screened hepatic transcription factors for efficient direct hepatic reprogramming and compared hepatic functions between hiHeps and other existing hepatocyte models. We found that human fibroblasts were efficiently converted into hiHeps by using a combination of ATF5, PROX1, FOXA2, FOXA3, and HNF4A (albumin+/alpha-1 antitrypsin+ cells = 27%, asialoglycoprotein receptor 1+ cells = 22%). The CYP expression levels and CYP activities in hiHeps were higher than those in human iPS cell-derived hepatocyte-like cells, but lower than those in short-term (4 hr) cultured primary human hepatocytes and primary human hepatocytes collected immediately after thawing. These results suggested that functional hiHeps could be efficiently generated by ATF5, PROX1, FOXA2, FOXA3, and HNF4A transduction. We believe that hiHeps generated by our method will be useful for the drug-discovery activities such as hepatotoxicity screening and drug metabolism tests.

Hepatitis C virus (HCV) infection is a major cause of liver-related morbidity and mortality. In order to develop effective remedies for hepatitis C, it is important to understand the HCV infection profile and host-HCV interaction. HCV-induced innate immune responses play a crucial role in spontaneous HCV clearance; however, HCV-induced innate immune responses have not been fully evaluated in hepatocytes, partly because there are few in vitro models of HCV-induced innate immunity. Recently, human induced pluripotent stem (iPS) cells have received much attention as an in vitro model of infection with various pathogens, including HCV. We previously established highly functional hepatocyte-like cells differentiated from human iPS cells (iPS-HLCs). Here, we examined the potential of iPS-HLCs as an in vitro HCV infection model, especially for evaluation of the relationship between HCV infection levels and HCV-induced innate immunity. Significant expressions of type I and III interferons (IFNs) and IFN-stimulated genes (ISGs) were induced following transfection with HCV genomic replicon RNA in iPS-HLCs. Following inoculation with the HCV JFH-1 strain in iPS-HLCs, peaks of HCV genome replication and HCV protein expression were observed on day 2, and then both the HCV genome and protein levels gradually declined, while the mRNA levels of type III IFNs and ISGs peaked at day 2 following inoculation. These results suggest that the HCV genome efficiently replicates in iPS-HLCs, resulting in HCV genome-induced up-regulation of IFNs and ISGs, and thereafter, HCV genome-induced up-regulation of IFNs and ISGs mediates a reduction in the HCV genome and protein levels in iPS-HLCs., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The function of hepatocytes largely depends on their position in the liver lobule. Although the method of differentiating hepatocytes from human pluripotent stem cells has been largely improved over the past decade, there remains no technique for generating hepatocyte-like cells (HLCs) with zone-specific hepatic properties. In this study, we searched for the factors that promote acquisition of zone-specific properties of HLCs. Here, we identified that WNT7B and WNT8B secreted from hepatocytes and cholangiocytes play important roles in achieving perivenous zone-specific characteristics, such as the enhancement of glutamine secretion, citric acid cycle, cytochrome P450 (CYP) 1A2 metabolism, and CYP1A2 induction capacities. We also found that WNT inhibitory factor (WIF-1) secreted from cholangiocytes was necessary for achieving periportal zone-specific characteristics, such as the enhancement of urea secretion and gluconeogenesis capacities. Therefore, WNT signal modulators secreted from hepatocytes or cholangiocytes conferred zone-specific hepatic properties onto HLCs., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

In order to understand the life cycle of hepatitis B virus (HBV) and to develop efficient anti-HBV drugs, a useful in vitro cell culture system which allows HBV infection and recapitulates virus-host interactions is essential; however, pre-existing in vitro HBV infection models are often problematic. Here, we examined the potential of human induced-pluripotent stem (iPS) cell-derived hepatocyte-like cells (iPS-HLCs) as an in vitro HBV infection model. Expression levels of several genes involved in HBV infection, including the sodium taurocholate cotransporting polypeptide (NTCP) gene, were gradually elevated as the differentiation status of human iPS cells proceeded to iPS-HLCs. The mRNA levels of these genes were comparable between primary human hepatocytes (PHHs) and iPS-HLCs. Following inoculation with HBV, we found significant production of HBV proteins and viral RNAs in iPS-HLCs. The three major forms of the HBV genome were detected in iPS-HLCs by Southern blotting analysis. Anti-HBV agents entecavir and Myrcludex-B, which are a nucleoside analogue reverse transcriptase inhibitor and a synthetic pre-S1 peptide, respectively, significantly inhibited HBV infection in iPS-HLCs. These data demonstrate that iPS-HLCs can be used as a promising in vitro HBV infection model.

Bile salt export pump (BSEP) plays an important role in hepatic secretion of bile acids and its deficiency results in severe cholestasis and liver failure. Mutation of the ABCB11 gene encoding BSEP induces BSEP deficiency and progressive familial intrahepatic cholestasis type 2 (PFIC2). Because liver transplantation remains standard treatment for PFIC2, the development of a novel therapeutic option is desired. However, a well reproducible model, which is essential for the new drug development for PFIC2, has not been established. Therefore, we attempted to establish a PFIC2 model by using iPSC technology. Human iPSCs were generated from patients with BSEP-deficiency (BD-iPSC), and were differentiated into hepatocyte-like cells (HLCs). In the BD-iPSC derived HLCs (BD-HLCs), BSEP was not expressed on the cell surface and the biliary excretion capacity was significantly impaired. We also identified a novel mutation in the 5'-untranslated region of the ABCB11 gene that led to aberrant RNA splicing in BD-HLCs. Furthermore, to evaluate the drug efficacy, BD-HLCs were treated with 4-phenylbutyrate (4PBA). The membrane BSEP expression level and the biliary excretion capacity in BD-HLCs were rescued by 4PBA treatment. In summary, we succeeded in establishing a PFIC2 model, which may be useful for its pathophysiological analysis and drug development., Competing Interests: The authors declare no competing financial interests.

Background & Aims: Hepatocyte transplantation is one of the most attractive approaches for the treatment of patients with liver failure. Because human induced pluripotent stem cell-derived hepatocyte-like cells (iPS-HLCs) can be produced on a large scale and generated from a patient with liver failure, they are expected to be used for hepatocyte transplantation. However, when using conventional transplantation methods, i.e., intrasplenic or portal venous infusion, it is difficult to control the engraftment efficiency and avoid unexpected engraftment in other organs because the transplanted cells are delivered into blood circulation before their liver engraftment., Methods: In this study, to resolve these issues, we attempted to employ a cell sheet engineering technology for experimental hepatocyte transplantation. The human iPS-HLC sheets were attached onto the liver surfaces of mice with liver injury., Results: This method reduced unexpected engraftment in organs other than the liver compared to that by intrasplenic transplantation. Human albumin levels in the mice with human iPS-HLC sheets were significantly higher than those in the intrasplenically-transplanted mice, suggesting the high potential for cell engraftment of the sheet transplantation procedure. In addition, human iPS-HLC sheet transplantation successfully ameliorated lethal acute liver injury induced by the infusion of carbon tetrachloride (CCl4). Moreover, we found that the hepatocyte growth factor secreted from the human iPS-HLC sheet played an important role in rescuing of mice from acute hepatic failure., Conclusions: Human iPS-HLC sheet transplantation would be a useful and reliable therapeutic approach for a patient with severe liver diseases., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatocyte-like cells differentiated from human iPS cells (human iPS-HLCs) are expected to be utilized in drug development and research. However, recent hepatic characterization of human iPS-HLCs showed that these cells resemble fetal hepatocytes rather than adult hepatocytes. Therefore, in this study, we aimed to develop a method to enhance the hepatic function of human iPS-HLCs. Because the gene expression levels of the hepatic transcription factors (activating transcription factor 5 (ATF5), CCAAT/enhancer-binding protein alpha (c/EBPα), and prospero homeobox protein 1 (PROX1)) in adult liver were significantly higher than those in human iPS-HLCs and fetal liver, we expected that the hepatic functions of human iPS-HLCs could be enhanced by adenovirus (Ad) vector-mediated ATF5, c/EBPα, and PROX1 transduction. The gene expression levels of cytochrome P450 (CYP) 2C9, 2E1, alpha-1 antitrypsin, transthyretin, Na+/taurocholate cotransporting polypeptide, and uridine diphosphate glucuronosyl transferase 1A1 and protein expression levels of CYP2C9 and CYP2E1 were upregulated by ATF5, c/EBPα, and PROX1 transduction. These results suggest that the hepatic functions of the human iPS-HLCs could be enhanced by ATF5, c/EBPα, and PROX1 transduction. Our findings would be useful for the hepatic maturation of human iPS-HLCs., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Human liver chimeric mice are expected to be applied for drug toxicity tests and human hepatitis virus research. Human induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs) are a highly attractive donor source for the generation of human liver chimeric mice because they can be produced on a large scale and established from an individual. Although these cells have been successfully used to generate human liver chimeric mice, there is still room for improvement in the repopulation efficiency. To enhance the repopulation efficacy, the human iPSC-HLCs were transduced with an adenovirus vector (Ad-FNK) expressing FNK, a hyperactive mutant gene from Bcl-xL, which was expected to inhibit apoptosis in the process of integration into liver parenchyma. We then transplanted Ad-FNK-transduced human iPSC-HLCs into urokinase-type plasminogen activator-transgenic severe combined immunodeficiency (uPA/SCID) mice (FNK mice) and evaluated the repopulation efficacy. The antiapoptotic effects of the human iPSC-HLCs were enhanced by FNK overexpression in vitro. Human albumin levels in the transplanted mice were significantly increased by transplantation of Ad-FNK-transduced human iPSC-HLCs (about 24,000 ng/ml). Immunohistochemical analysis with an anti-human αAT antibody revealed greater repopulation efficacy in the livers of FNK mice than control mice. Interestingly, the expression levels of human hepatocyte-related genes in the human iPSC-HLCs of FNK mice were much higher than those in the human iPSC-HLCs before transplantation. We succeeded in improving the repopulation efficacy of human liver chimeric mice generated by transplanting the Ad-FNK-transduced human iPSC-HLCs into uPA/SCID mice. Our method using ectopic expression of FNK was useful for generating human chimeric mice with high chimerism.

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.

Human embryonic stem cells (hESCs) and their derivatives are expected to be used in drug discovery, regenerative medicine and the study of human embryogenesis. Because hepatocyte differentiation from hESCs has the potential to recapitulate human liver development in vivo, we employed this differentiation method to investigate the molecular mechanisms underlying human hepatocyte differentiation. A previous study has shown that a gradient of transforming growth factor beta (TGFβ) signaling is required to segregate hepatocyte and cholangiocyte lineages from hepatoblasts. Although CCAAT/enhancer binding proteins (c/EBPs) are known to be important transcription factors in liver development, the relationship between TGFβ signaling and c/EBP-mediated transcriptional regulation in the hepatoblast fate decision is not well known. To clarify this relationship, we examined whether c/EBPs could determine the hepatoblast fate decision via regulation of TGFβ receptor 2 (TGFBR2) expression in the hepatoblast-like cells differentiated from hESCs. We found that TGFBR2 promoter activity was negatively regulated by c/EBPα and positively regulated by c/EBPβ. Moreover, c/EBPα overexpression could promote hepatocyte differentiation by suppressing TGFBR2 expression, whereas c/EBPβ overexpression could promote cholangiocyte differentiation by enhancing TGFBR2 expression. Our findings demonstrated that c/EBPα and c/EBPβ determine the lineage commitment of hepatoblasts by negatively and positively regulating the expression of a common target gene, TGFBR2, respectively.

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background & Aims: Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can be utilized as a tool for screening for hepatotoxicity in the early phase of pharmaceutical development. We have recently reported that hepatic differentiation is promoted by sequential transduction of SOX17, HEX, and HNF4α into hESC- or hiPSC-derived cells, but further maturation of hepatocyte-like cells is required for widespread use of drug screening., Methods: To screen for hepatic differentiation-promoting factors, we tested the seven candidate genes related to liver development., Results: The combination of two transcription factors, FOXA2 and HNF1α, promoted efficient hepatic differentiation from hESCs and hiPSCs. The expression profile of hepatocyte-related genes (such as genes encoding cytochrome P450 enzymes, conjugating enzymes, hepatic transporters, and hepatic nuclear receptors) achieved with FOXA2 and HNF1α transduction was comparable to that obtained in primary human hepatocytes. The hepatocyte-like cells generated by FOXA2 and HNF1α transduction exerted various hepatocyte functions including albumin and urea secretion, and the uptake of indocyanine green and low density lipoprotein. Moreover, these cells had the capacity to metabolize all nine tested drugs and were successfully employed to evaluate drug-induced cytotoxicity., Conclusions: Our method employing the transduction of FOXA2 and HNF1α represents a useful tool for the efficient generation of metabolically functional hepatocytes from hESCs and hiPSCs, and the screening of drug-induced cytotoxicity., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity.

Host tropism of hepatitis C virus (HCV) is limited to human and chimpanzee. HCV infection has never been fully understood because there are few conventional models for HCV infection. Human induced pluripotent stem cell-derived hepatocyte-like (iPS-Hep) cells have been expected to use for drug discovery to predict therapeutic activities and side effects of compounds during the drug discovery process. However, the suitability of iPS-Hep cells as an experimental model for HCV research is not known. Here, we investigated the entry and genomic replication of HCV in iPS-Hep cells by using HCV pseudotype virus (HCVpv) and HCV subgenomic replicons, respectively. We showed that iPS-Hep cells, but not iPS cells, were susceptible to infection with HCVpv. The iPS-Hep cells expressed HCV receptors, including CD81, scavenger receptor class B type I (SR-BI), claudin-1, and occludin; in contrast, the iPS cells showed no expression of SR-BI or claudin-1. HCV RNA genome replication occurred in the iPS-Hep cells. Anti-CD81 antibody, an inhibitor of HCV entry, and interferon, an inhibitor of HCV genomic replication, dose-dependently attenuated HCVpv entry and HCV subgenomic replication in iPS-Hep cells, respectively. These findings suggest that iPS-Hep cells are an appropriate model for HCV infection., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.