Liver regeneration and inflammation: from fundamental science to clinical applications.

Liver regeneration is a complex process involving the crosstalk of multiple cell types, including hepatocytes, hepatic stellate cells, endothelial cells and inflammatory cells. The healthy liver is mitotically quiescent, but following toxic damage or resection the cells can rapidly enter the cell cycle to restore liver mass and function. During this process of regeneration, epithelial and non-parenchymal cells respond in a tightly coordinated fashion. Recent studies have described the interaction between inflammatory cells and a number of other cell types in the liver. In particular, macrophages can support biliary regeneration, contribute to fibrosis remodelling by repressing hepatic stellate cell activation and improve liver regeneration by scavenging dead or dying cells in situ. In this Review, we describe the mechanisms of tissue repair following damage, highlighting the close relationship between inflammation and liver regeneration, and discuss how recent findings can help design novel therapeutic approaches.

Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture.

In the healthy adult liver, most hepatocytes proliferate minimally. However, upon physical or chemical injury to the liver, hepatocytes proliferate extensively in vivo under the direction of multiple extracellular cues, including Wnt and pro-inflammatory signals. Currently, liver organoids can be generated readily in vitro from bile-duct epithelial cells, but not hepatocytes. Here, we show that TNFα, an injury-induced inflammatory cytokine, promotes the expansion of hepatocytes in 3D culture and enables serial passaging and long-term culture for more than 6 months. Single-cell RNA sequencing reveals broad expression of hepatocyte markers. Strikingly, in vitro-expanded hepatocytes engrafted, and significantly repopulated, the injured livers of Fah-/- mice. We anticipate that tissue repair signals can be harnessed to promote the expansion of otherwise hard-to-culture cell-types, with broad implications.

Hybrid Periportal Hepatocytes Regenerate the Injured Liver without Giving Rise to Cancer.

Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.

The RNA sensor RIG-I dually functions as an innate sensor and direct antiviral factor for hepatitis B virus.

Host innate recognition triggers key immune responses for viral elimination. The sensing mechanism of hepatitis B virus (HBV), a DNA virus, and the subsequent downstream signaling events remain to be fully clarified. Here we found that type III but not type I interferons are predominantly induced in human primary hepatocytes in response to HBV infection, through retinoic acid-inducible gene-I (RIG-I)-mediated sensing of the 5'-ε region of HBV pregenomic RNA. In addition, RIG-I could also counteract the interaction of HBV polymerase (P protein) with the 5'-ε region in an RNA-binding dependent manner, which consistently suppressed viral replication. Liposome-mediated delivery and vector-based expression of this ε region-derived RNA in liver abolished the HBV replication in human hepatocyte-chimeric mice. These findings identify an innate-recognition mechanism by which RIG-I dually functions as an HBV sensor activating innate signaling and to counteract viral polymerase in human hepatocytes.

Irreversible Electroporation of the Liver Increases the Transplant Engraftment of Hepatocytes.

INTRODUCTION: Irreversible electroporation (IRE) is a tissue ablation technology that kills cells with short electrical pulses that do not induce thermal damage, thereby preserving the extracellular matrix. Preclinical research suggests that IRE may be developed as a tool for regenerative surgery by clearing existing host cells within a solid organ and creating a supportive niche for new cell engraftment. We hypothesized that hepatocytes transplanted by injection into the portal circulation would preferentially engraft within liver parenchyma pretreated with IRE. METHODS: Transgene-positive ß-galactosidase-expressing hepatocytes were isolated from B6.129S7-Gt(ROSA)26Sor/J (ROSA26) mice and transplanted by intrasplenic injection into wild-type littermates that received liver IRE pretreatment or control sham treatment. Engraftment of donor hepatocytes in recipient livers was determined by X-gal staining. RESULTS: Significantly higher numbers of X-gal+ donor hepatocytes engrafted in the livers of IRE-treated mice as compared to sham-treated mice. X-gal+ hepatocytes persisted in IRE-treated recipients for at least 11 d post-transplant and formed clusters. Immunostaining demonstrated the presence of HNF4A/Ki67/ß-galactosidase triple-positive cells within IRE-ablation zones, indicating that transplanted hepatocytes preferentially engrafted in IRE-treated liver parenchyma and proliferated. CONCLUSIONS: IRE pretreatment of the liver increased engraftment of transplanted hepatocytes within the IRE-ablation zone. IRE treatment of the host liver may be developed clinically as a strategy to increase engraftment efficiency of primary hepatocytes and/or hepatocytes derived from stem cells in cell transplant therapies.

Viability assessment of the liver during ex-situ machine perfusion prior to transplantation.

PURPOSE OF REVIEW: In an attempt to reduce waiting list mortality in liver transplantation, less-than-ideal quality donor livers from extended criteria donors are increasingly accepted. Predicting the outcome of these organs remains a challenge. Machine perfusion provides the unique possibility to assess donor liver viability pretransplantation and predict postreperfusion organ function. RECENT FINDINGS: Assessing liver viability during hypothermic machine perfusion remains challenging, as the liver is not metabolically active. Nevertheless, the levels of flavin mononucleotide, transaminases, lactate dehydrogenase, glucose and pH in the perfusate have proven to be predictors of liver viability. During normothermic machine perfusion, the liver is metabolically active and in addition to the perfusate levels of pH, transaminases, glucose and lactate, the production of bile is a crucial criterion for hepatocyte viability. Cholangiocyte viability can be determined by analyzing bile composition. The differences between perfusate and bile levels of pH, bicarbonate and glucose are good predictors of freedom from ischemic cholangiopathy. SUMMARY: Although consensus is lacking regarding precise cut-off values during machine perfusion, there is general consensus on the importance of evaluating both hepatocyte and cholangiocyte compartments. The challenge is to reach consensus for increased organ utilization, while at the same time pushing the boundaries by expanding the possibilities for viability testing.

Consumptive coagulopathy of severe yellow fever occurs independently of hepatocellular tropism and massive hepatic injury.

Yellow fever (YF) is a mosquito-transmitted viral disease that causes tens of thousands of deaths each year despite the long-standing deployment of an effective vaccine. In its most severe form, YF manifests as a hemorrhagic fever that causes severe damage to visceral organs. Although coagulopathy is a defining feature of severe YF in humans, the mechanism by which it develops remains uncertain. Hepatocytes are a major target of yellow fever virus (YFV) infection, and the coagulopathy in severe YF has long been attributed to massive hepatocyte infection and destruction that results in a defect in clotting factor synthesis. However, when we analyzed blood from Brazilian patients with severe YF, we found high concentrations of plasma D-dimer, a fibrin split product, suggestive of a concurrent consumptive process. To define the relationship between coagulopathy and hepatocellular tropism, we compared infection and disease in Fah-/-, Rag2-/-, and Il2rɣ-/- mice engrafted with human hepatocytes (hFRG mice) and rhesus macaques using a highly pathogenic African YFV strain. YFV infection of macaques and hFRG mice caused substantial hepatocyte infection, liver damage, and coagulopathy as defined by virological, clinical, and pathological criteria. However, only macaques developed a consumptive coagulopathy whereas YFV-infected hFRG mice did not. Thus, infection of cell types other than hepatocytes likely contributes to the consumptive coagulopathy associated with severe YF in primates and humans. These findings expand our understanding of viral hemorrhagic disease and associated coagulopathy and suggest directions for clinical management of severe YF cases.

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes.

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

Magnetic Nanostructures and Stem Cells for Regenerative Medicine, Application in Liver Diseases.

The term "liver disease" refers to any hepatic condition that leads to tissue damage or altered hepatic function and can be induced by virus infections, autoimmunity, inherited genetic mutations, high consumption of alcohol or drugs, fat accumulation, and cancer. Some types of liver diseases are becoming more frequent worldwide. This can be related to increasing rates of obesity in developed countries, diet changes, higher alcohol intake, and even the coronavirus disease 2019 (COVID-19) pandemic was associated with increased liver disease-related deaths. Although the liver can regenerate, in cases of chronic damage or extensive fibrosis, the recovery of tissue mass is impossible, and a liver transplant is indicated. Because of reduced organ availability, it is necessary to search for alternative bioengineered solutions aiming for a cure or increased life expectancy while a transplant is not possible. Therefore, several groups were studying the possibility of stem cells transplantation as a therapeutic alternative since it is a promising strategy in regenerative medicine for treating various diseases. At the same time, nanotechnological advances can contribute to specifically targeting transplanted cells to injured sites using magnetic nanoparticles. In this review, we summarize multiple magnetic nanostructure-based strategies that are promising for treating liver diseases.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

Domino hepatocyte transplantation using explanted human livers with metabolic defects attenuates D-GalN/LPS-induced acute liver failure.

BACKGROUND: Explanted livers from patients with inherited metabolic liver diseases possess the potential to be a cell source of good-quality hepatocytes for hepatocyte transplantation (HT). This study evaluated the therapeutic effects of domino HT using hepatocytes isolated from explanted human livers for acute liver failure (ALF). METHODS: Isolated hepatocytes were evaluated for viability and function and then transplanted into D-galactosamine/lipopolysaccharide-induced ALF mice via splenic injection. The survival rate was analyzed by the Kaplan-Meier method and log-rank test. Liver function was evaluated by serum biochemical parameters, and inflammatory cytokine levels were measured by ELISA. The pathological changes in the liver tissues were assessed by hematoxylin-eosin staining. Hepatocyte apoptosis was investigated by TUNEL, and hepatocyte apoptosis-related proteins were detected by western blot. The localization of human hepatocytes in the injured mouse livers was detected by immunohistochemical analyses. RESULTS: Hepatocytes were successfully isolated from explanted livers of 10 pediatric patients with various liver-based metabolic disorders, with an average viability of 85.3% ± 13.0% and average yield of 9.2 × 106 ± 3.4 × 106 cells/g. Isolated hepatocytes had an excellent ability to secret albumin, produce urea, uptake indocyanine green, storage glycogen, and express alpha 1 antitrypsin, albumin, cytokeratin 18, and CYP3A4. Domino HT significantly reduced mortality, decreased serum levels of alanine aminotransferase and aspartate aminotransferase, and improved the pathological damage. Moreover, transplanted hepatocytes inhibited interleukin-6 and tumor necrosis factor-α levels. Domino HT also ameliorates hepatocyte apoptosis, as evidenced by decreased TUNEL positive cells. Positive staining for human albumin suggested the localization of human hepatocytes in ALF mice livers. CONCLUSION: Explanted livers from patients with inheritable metabolic disorders can serve as a viable cell source for cell-based therapies. Domino HT using hepatocytes with certain metabolic defects has the potential to be a novel therapeutic strategy for ALF.

Tumor Necrosis Factor Directs Allograft-Related Innate Responses and Its Neutralization Improves Hepatocyte Engraftment in Rats.

The innate immune system plays a critical role in allograft rejection. Alloresponses involve numerous cytokines, chemokines, and receptors that cause tissue injury during rejection. To dissect these inflammatory mechanisms, we developed cell transplantation models in dipeptidylpeptidase-deficient F344 rats using mycophenolate mofetil and tacrolimus for partial lymphocyte-directed immunosuppression. Syngeneic hepatocytes engrafted in liver, whereas allogeneic hepatocytes were rejected but engrafted after immunosuppression. These transplants induced mRNAs for >40 to 50 cytokines, chemokines, and receptors. In allografts, innate cell type-related regulatory networks extended to granulocytes, monocytes, and macrophages. Activation of Tnfa and its receptors or major chemokine receptor-ligand subsets persisted in the long term. An examination of the contribution of Tnfa in allograft response revealed that it was prospectively antagonized by etanercept or thalidomide, which resolved cytokine, chemokine, and receptor cascades. In bioinformatics analysis of upstream regulator networks, the Cxcl8 pathway exhibited dominance despite immunosuppression. Significantly, Tnfa antagonism silenced the Cxcl8 pathway and decreased neutrophil and Kupffer cell recruitment, resulting in multifold greater engraftment of allogeneic hepatocytes and substantially increased liver repopulation in retrorsine/partial hepatectomy model. We conclude that Tnfa is a major driver for persistent innate immune responses after allogeneic cells. Neutralizing Tnfa should help in avoiding rejection and associated tissue injury in the allograft setting.

Advanced Techniques and Awaited Clinical Applications for Human Pluripotent Stem Cell Differentiation into Hepatocytes.

Liver transplantation is currently the only curative treatment for several liver diseases such as acute liver failure, end-stage liver disorders, primary liver cancers, and certain genetic conditions. Unfortunately, despite improvements to transplantation techniques, including live donor transplantation, the number of organs available remains insufficient to meet patient needs. Hepatocyte transplantation has enabled some encouraging results as an alternative to organ transplantation, but primary hepatocytes are little available and cannot be amplified using traditional two-dimensional culture systems. Indeed, although recent studies have tended to show that three-dimensional culture enables long-term hepatocyte culture, it is still agreed that, like most adult primary cell types, hepatocytes remain refractory to in vitro expansion. Because of their exceptional properties, human pluripotent stem cells (hPSCs) can be amplified indefinitely and differentiated into any cell type, including liver cells. While many teams have worked on hepatocyte differentiation, there has been a consensus that cells obtained after hPSC differentiation have more fetal than adult hepatocyte characteristics. New technologies have been used to improve the differentiation process in recent years. This review discusses the technical improvements made to hepatocyte differentiation protocols and the clinical approaches developed to date and anticipated in the near future.

Dynamic Transcriptional and Epigenetic Changes Drive Cellular Plasticity in the Liver.

BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.

Functional Comparison of Interferon-α Subtypes Reveals Potent Hepatitis B Virus Suppression by a Concerted Action of Interferon-α and Interferon-γ Signaling.

BACKGROUND AND AIMS: Interferon (IFN)-α, composed of numerous subtypes, plays a crucial role in immune defense. As the most studied subtype, IFN-α2 has been used for treating chronic hepatitis B virus (HBV) infection, with advantages of finite treatment duration and sustained virologic response, but its efficacy remains relatively low. This study aimed to screen for IFN-α subtypes with the highest anti-HBV potency and to characterize mechanisms of IFN-α-mediated HBV restriction. APPROACH AND RESULTS: Using cell culture-based HBV infection systems and a human-liver chimeric mouse model, IFN-α subtype-mediated antiviral response and signaling activation were comprehensively analyzed. IFN-α14 was identified as the most effective subtype in suppression of HBV covalently closed circular DNA transcription and HBV e antigen/HBV surface antigen production, with median inhibitory concentration values approximately 100-fold lower than those of the conventional IFN-α2. IFN-α14 alone elicited IFN-α and IFN-γ signaling crosstalk in a manner similar to the combined use of IFN-α2 and IFN-γ, inducing multiple potent antiviral effectors, which synergistically restricted HBV replication. Guanylate binding protein 5, one of the most differentially expressed genes between IFN-α14-treated and IFN-α2-treated liver cells, was identified as an HBV restriction factor. A strong IFN-α-IFN-α receptor subunit 1 interaction determines the anti-HBV activity of IFN-α. The in vivo anti-HBV activity of IFN-α14 and treatment-related transcriptional patterns were further confirmed, and few adverse effects were observed. CONCLUSIONS: A concerted IFN-α and IFN-γ response in liver, which could be efficiently elicited by IFN-α subtype 14, is associated with potent HBV suppression. These data deepen the understanding of the divergent activities of IFN-α subtypes and the mechanism underlying the synergism between IFN-α and IFN-γ signaling, with implications for improved IFN therapy and HBV curative strategies.

Clinical Implications of HBV PreS/S Mutations and the Effects of PreS2 Deletion on Mitochondria, Liver Fibrosis, and Cancer Development.

BACKGROUND AND AIMS: PreS mutants of HBV have been reported to be associated with HCC. We conducted a longitudinal study of the role of HBV preS mutations in the development of HCC, particularly in patients with chronic hepatitis B (CHB) having low HBV DNA or alanine aminotransferase (ALT) levels, and investigated the effects of secretion-defective preS2 deletion mutant (preS2ΔMT) on hepatocyte damage in vitro and liver fibrosis in vivo. APPROACH AND RESULTS: Association of preS mutations with HCC in 343 patients with CHB was evaluated by a retrospective case-control follow-up study. Effects of preS2ΔMT on HBsAg retention, endoplasmic reticulum (ER) stress, calcium accumulation, mitochondrial dysfunction, and liver fibrosis were examined. Multivariate analysis revealed a significant association of preS mutations with HCC (HR, 3.210; 95% CI, 1.072-9.613; P = 0.037) including cases with low HBV DNA or ALT levels (HR, 2.790; 95% CI, 1.133-6.873; P = 0.026). Antiviral therapy reduced HCC risk, including cases with preS mutations. PreS2ΔMT expression promoted HBsAg retention in the ER and unfolded protein response (UPR). Transmission electron microscopic examination, MitoTracker staining, real-time ATP assay, and calcium staining of preS2ΔMT-expressing cells revealed aberrant ER and mitochondrial ultrastructure, reduction of mitochondrial membrane potential and ATP production, and calcium overload. Serum HBV secretion levels were ~100-fold lower in preS2ΔMT-infected humanized Fah-/-/ Rag2-/-/Il2rg-/- triple knockout mice than in wild-type HBV-infected mice. PreS2ΔMT-infected mice displayed up-regulation of UPR and caspase-3 and enhanced liver fibrosis. CONCLUSIONS: PreS mutations were significantly associated with HCC development in patients with CHB, including those with low HBV DNA or ALT levels. Antiviral therapy reduced HCC occurrence in patients with CHB, including those with preS mutations. Intracellular accumulation of mutated HBsAg induced or promoted ER stress, calcium overload, mitochondrial dysfunction, impaired energy metabolism, liver fibrosis, and HCC.

Ethanol attenuates presentation of cytotoxic T-lymphocyte epitopes on hepatocytes of HBV-infected humanized mice.

BACKGROUND AND AIMS: Approximately 3.5% of the global population is chronically infected with Hepatitis B Virus (HBV), which puts them at high risk of end-stage liver disease, with the risk of persistent infection potentiated by alcohol consumption. However, the mechanisms underlying the effects of alcohol on HBV persistence remain unclear. Here, we aimed to establish in vivo/ex vivo evidence that alcohol suppresses HBV peptides-major histocompatibility complex (MHC) class I antigen display on primary human hepatocytes (PHH), which diminishes the recognition and clearance of HBV-infected hepatocytes by cytotoxic T-lymphocytes (CTLs). METHODS: We used fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knock-out (FRG-KO) humanized mice transplanted with human leukocyte antigen-A2 (HLA-A2)-positive hepatocytes. The mice were HBV-infected and fed control and alcohol diets. Isolated hepatocytes were exposed ex vivo to HBV 18-27-HLA-A2-restricted CTLs to quantify cytotoxicity. For mechanistic studies, we measured proteasome activities, unfolded protein response (UPR), and endoplasmic reticulum (ER) stress in hepatocytes from HBV-infected humanized mouse livers. RESULTS AND CONCLUSIONS: We found that alcohol feeding attenuated HBV core 18-27-HLA-A2 complex presentation on infected hepatocytes due to the suppression of proteasome function and ER stress induction, which diminished both the processing of HBV peptides and trafficking of HBV-MHC class I complexes to the hepatocyte surface. This alcohol-mediated decrease in MHC class I-restricted antigen presentation of the CTL epitope on target hepatocytes reduced the CTL-specific elimination of infected cells, potentially leading to HBV-infection persistence, which promotes end-stage liver disease outcomes.

AAV integration in human hepatocytes.

Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes.

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Insights From Liver-Humanized Mice on Cholesterol Lipoprotein Metabolism and LXR-Agonist Pharmacodynamics in Humans.

BACKGROUND AND AIMS: Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver-humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species-related, physiological differences. APPROACH AND RESULTS: Fah-/- , Rag2-/- , and Il2rg-/- knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human-like pattern, characterized by a high ratio of low-density lipoprotein to high-density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low-density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human-like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. CONCLUSIONS: LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.