Hepatitis E virus RNA-dependent RNA polymerase is involved in RNA replication and infectious particle production.

BACKGROUND AND AIMS: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis worldwide. Its positive-strand RNA genome encodes three open reading frames (ORF). ORF1 is translated into a large protein composed of multiple domains and is known as the viral replicase. The RNA-dependent RNA polymerase (RDRP) domain is responsible for the synthesis of viral RNA. APPROACH AND RESULTS: Here, we identified a highly conserved α-helix located in the RDRP thumb subdomain. Nuclear magnetic resonance demonstrated an amphipathic α-helix extending from amino acids 1628 to 1644 of the ORF1 protein. Functional analyses revealed a dual role of this helix in HEV RNA replication and virus production, including assembly and release. Mutations on the hydrophobic side of the amphipathic α-helix impaired RNA replication and resulted in the selection of a second-site compensatory change in the RDRP palm subdomain. Other mutations enhanced RNA replication but impaired virus assembly and/or release. CONCLUSIONS: Structure-function analyses identified a conserved amphipathic α-helix in the thumb subdomain of the HEV RDRP with a dual role in viral RNA replication and infectious particle production. This study provides structural insights into a key segment of the ORF1 protein and describes the successful use of reverse genetics in HEV, revealing functional interactions between the RDRP thumb and palm subdomains. On a broader scale, it demonstrates that the HEV replicase, similar to those of other positive-strand RNA viruses, is also involved in virus production.

Recombinant Hepatitis E Viruses Harboring Tags in the ORF1 Protein.

Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.

Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion.

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.

Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine.

The liver is one of the major organs in the human body. Severe or prolonged exposure of the liver to different factors may cause life-threatening disease, which necessitates donor organ transplantation. While orthotopic liver transplantation can be used to effectively treat liver failure, it is an invasive procedure, which is severely limited by organ donation. Therefore, alternative sources of liver support have been proposed and studied. This includes the use of pluripotent stem cell-derived hepatocytes as a renewable source of cells for therapy. In addition to cell-based therapies, in vitro engineered liver tissue provides powerful models for human drug discovery and disease modeling. This review focuses on the generation of hepatocyte-like cells from pluripotent stem cells and their application in translational medicine. Stem Cells 2016;34:1421-1426.

Pluripotent stem cell-derived hepatocytes: potential and challenges in pharmacology.

The liver is a fascinating organ and performs a wide range of functions necessary for life. Because the hepatocyte is the major functional cell type found in the liver, it is important that we better understand its role in health and disease. Functional hepatocytes have been derived from many sources, including human stem cell populations. These models offer new opportunities to further our understanding of human liver biology from diverse genotypes and, in the future, to facilitate the development of novel medicines or cell-based therapies. This review discusses limitations in current cell-based models and the advantages offered by pluripotent stem cell-derived hepatocytes.

Fluid shear stress modulation of hepatocyte-like cell function.

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype.

Gene networks and transcription factor motifs defining the differentiation of stem cells into hepatocyte-like cells.

BACKGROUND & AIMS: The differentiation of stem cells to hepatocyte-like cells (HLC) offers the perspective of unlimited supply of human hepatocytes. However, the degree of differentiation of HLC remains controversial. To obtain an unbiased characterization, we performed a transcriptomic study with HLC derived from human embryonic and induced stem cells (ESC, hiPSC) from three different laboratories. METHODS: Genome-wide gene expression profiles of ESC and HLC were compared to freshly isolated and up to 14days cultivated primary human hepatocytes. Gene networks representing successful and failed hepatocyte differentiation, and the transcription factors involved in their regulation were identified. RESULTS: Gene regulatory network analysis demonstrated that HLC represent a mixed cell type with features of liver, intestine, fibroblast and stem cells. The "unwanted" intestinal features were associated with KLF5 and CDX2 transcriptional networks. Cluster analysis identified highly correlated groups of genes associated with mature liver functions (n=1057) and downregulated proliferation associated genes (n=1562) that approach levels of primary hepatocytes. However, three further clusters containing 447, 101, and 505 genes failed to reach levels of hepatocytes. Key TF of two of these clusters include SOX11, FOXQ1, and YBX3. The third unsuccessful cluster, controlled by HNF1, CAR, FXR, and PXR, strongly overlaps with genes repressed in cultivated hepatocytes compared to freshly isolated hepatocytes, suggesting that current in vitro conditions lack stimuli required to maintain gene expression in hepatocytes, which consequently also explains a corresponding deficiency of HLC. CONCLUSIONS: The present gene regulatory network approach identifies key transcription factors which require modulation to improve HLC differentiation.

Liver biopsy derived induced pluripotent stem cells provide unlimited supply for the generation of hepatocyte-like cells.

BACKGROUND & AIMS: Hepatocyte-like cells (HLCs) differentiated from induced pluripotent stem cells (iPSCs) have emerged as a promising cell culture model to study metabolism, biotransformation, viral infections and inherited liver diseases. iPSCs provide an unlimited supply for the generation of HLCs, but incomplete HLC differentiation remains a major challenge. iPSC may carry-on a tissue of origin dependent expression memory influencing iPSC differentiation into different cell types. Whether liver derived iPSCs (Li-iPSCs) would allow the generation of more fully differentiated HLCs is not known. METHODS: In the current study, we used primary liver cells (PLCs) expanded from liver needle biopsies and reprogrammed them into Li-iPSCs using a non-integrative Sendai virus-based system. Li-iPSCs were differentiated into HLCs using established differentiation protocols. The HLC phenotype was characterized at the protein, functional and transcriptional level. RNA sequencing data were generated from the originating liver biopsies, the Li-iPSCs, fibroblast derived iPSCs, and differentiated HLCs, and used to characterize and compare their transcriptome profiles. RESULTS: Li-iPSCs indeed retain a liver specific transcriptional footprint. Li-iPSCs can be propagated to provide an unlimited supply of cells for differentiation into Li-HLCs. Similar to HLCs derived from fibroblasts, Li-HLCs could not be fully differentiated into hepatocytes. Relative to the originating liver, Li-HLCs showed lower expression of liver specific transcription factors and increased expression of genes involved in the differentiation of other tissues. CONCLUSIONS: PLCs and Li-iPSCs obtained from small pieces of human needle liver biopsies constitute a novel unlimited source for the production of HLCs. Despite the preservation of a liver specific gene expression footprint in Li-iPSCs, the generation of fully differentiated hepatocytes cannot be achieved with the current differentiation protocols.

Modelling non-alcoholic fatty liver disease in human hepatocyte-like cells.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in developed countries. An in vitro NAFLD model would permit mechanistic studies and enable high-throughput therapeutic screening. While hepatic cancer-derived cell lines are a convenient, renewable resource, their genomic, epigenomic and functional alterations mean their utility in NAFLD modelling is unclear. Additionally, the epigenetic mark 5-hydroxymethylcytosine (5hmC), a cell lineage identifier, is rapidly lost during cell culture, alongside expression of the Ten-eleven-translocation (TET) methylcytosine dioxygenase enzymes, restricting meaningful epigenetic analysis. Hepatocyte-like cells (HLCs) derived from human embryonic stem cells can provide a non-neoplastic, renewable model for liver research. Here, we have developed a model of NAFLD using HLCs exposed to lactate, pyruvate and octanoic acid (LPO) that bear all the hallmarks, including 5hmC profiles, of liver functionality. We exposed HLCs to LPO for 48 h to induce lipid accumulation. We characterized the transcriptome using RNA-seq, the metabolome using ultra-performance liquid chromatography-mass spectrometry and the epigenome using 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) sequencing. LPO exposure induced an NAFLD phenotype in HLCs with transcriptional and metabolomic dysregulation consistent with those present in human NAFLD. HLCs maintain expression of the TET enzymes and have a liver-like epigenome. LPO exposure-induced 5hmC enrichment at lipid synthesis and transport genes. HLCs treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and additionally retain TET expression and 5hmC. This in vitro model of NAFLD will be useful for future mechanistic and therapeutic studies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Reducing Hepatocyte Injury and Necrosis in Response to Paracetamol Using Noncoding RNAs.

UNLABELLED: The liver performs multiple functions within the human body. It is composed of numerous cell types, which play important roles in organ physiology. Our study centers on the major metabolic cell type of the liver, the hepatocyte, and its susceptibility to damage during drug overdose. In these studies, hepatocytes were generated from a renewable and genetically defined resource. In vitro-derived hepatocytes were extensively profiled and exposed to varying levels of paracetamol and plasma isolated from liver-failure patients, with a view to identifying noncoding microRNAs that could reduce drug- or serum-induced hepatotoxicity. We identified a novel anti-microRNA, which reduced paracetamol-induced hepatotoxicity and glutathione depletion. Additionally, we identified a prosurvival role for anti-microRNA-324 following exposure to plasma collected from liver failure patients. We believe that these studies represent an important advance for the field, demonstrating the power of stem cell-derived systems to model human biology "in a dish" and identify novel noncoding microRNAs, which could be translated to the clinic in the future. SIGNIFICANCE: The liver performs vital functions within the human body and is composed of numerous cell types. The major metabolic cell type of the liver, the hepatocyte, is susceptible to damage during drug overdose. In these studies, hepatocytes were generated from a renewable resource and exposed to varying levels of paracetamol, with a view to identifying interventions that could reduce or attenuate drug-induced liver toxicity. A novel noncoding RNA that reduced paracetamol-induced hepatocyte toxicity was identified. These findings may represent an important advance for the field.

Serum-Free Directed Differentiation of Human Embryonic Stem Cells to Hepatocytes.

The increase in human liver disease worldwide is a major concern. At present, the only successful mode of treatment for failing liver function is organ transplantation. While highly successful, donor organs are a limited resource that cannot meet current demands. Therefore, alternative liver support strategies have been explored, including the use of the major and metabolic cell within the liver, the hepatocyte. While current approaches using human hepatocytes are very promising, donor material is still required and therefore suffers from similar limitations to whole organ transplantation. One alternative source of human hepatocytes being actively pursued in the field is pluripotent stem cells. Pluripotent stem cells are a scalable and renewable cell-based resource, which can be efficiently differentiated towards hepatocytes, including pluripotent stem cell lines that have been derived under good manufacturing practice conditions. Therefore, it is believed that this approach provides a promising model system for cell scale-up and differentiation. In the future, pluripotent stem cell-derived hepatocytes could be used in the clinic to support failing liver function if they should be deemed fit for purpose.

Polyurethane: Stable Cell Phenotype Requires Plasticity: Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance (Adv. Healthcare Mater. 12/2015).

One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. On page 1820 David C. Hay and co-workers describe the use of a synthetic polymer surface. The image shows stem cell-derived hepatocytes replated on this polyurethane surface. Importantly the cells express Zonal Occludin (green stain) at the cell surface, which indicates that the cells display elements of polarization. The blue stain is DAPI, which demarks the nucleus.

Polymer Supported Directed Differentiation Reveals a Unique Gene Signature Predicting Stable Hepatocyte Performance.

In theory, pluripotent stem cells can give rise to all somatic cell types found in the human body. The ability to generate renewable sources of human cells has enormous potential to improve human health and wealth. One major obstacle to the routine deployment of stem cell-derived cells is their instability in culture. To tackle this issue a synthetic polymer surface is used.

Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

Deriving functional hepatocytes from pluripotent stem cells.

Despite major progress in the management of human liver disease, the only cure for a critically failing organ is liver transplantation. While a highly successful approach, the use of cadaveric organs as a routine treatment option is severely limited by organ availability. Therefore, the use of cell-based therapies has been explored to provide support for the failing liver. In addition to developing new treatments, there is also an imperative to develop better human models 'in a dish'. Such approaches will undoubtedly lead to a better understanding of the disease process, offering new treatment or preventative strategies. With both approaches in mind, we have developed robust hepatocyte differentiation methodologies for use with pluripotent stem cells. Importantly, our procedure is highly efficient (∼ 90%) and delivers active, drug-inducible, and predictive human hepatocyte populations.

Modulating innate immunity improves hepatitis C virus infection and replication in stem cell-derived hepatocytes.

In this study, human embryonic stem cell-derived hepatocytes (hESC-Heps) were investigated for their ability to support hepatitis C virus (HCV) infection and replication. hESC-Heps were capable of supporting the full viral life cycle, including the release of infectious virions. Although supportive, hESC-Hep viral infection levels were not as great as those observed in Huh7 cells. We reasoned that innate immune responses in hESC-Heps may lead to the low level of infection and replication. Upon further investigation, we identified a strong type III interferon response in hESC-Heps that was triggered by HCV. Interestingly, specific inhibition of the JAK/STAT signaling pathway led to an increase in HCV infection and replication in hESC-Heps. Of note, the interferon response was not evident in Huh7 cells. In summary, we have established a robust cell-based system that allows the in-depth study of virus-host interactions in vitro.

Stabilizing hepatocellular phenotype using optimized synthetic surfaces.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

Accurate prediction of drug-induced liver injury using stem cell-derived populations.

Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies.