Acquisition of epithelial plasticity in human chronic liver disease.

For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.

Convergent somatic mutations in metabolism genes in chronic liver disease.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes1-8. Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease9-13 than in normal liver13-16, which enables positive selection to shape the genomic landscape9-13. Here we analysed somatic mutations from 1,590 genomes across 34 liver samples, including healthy controls, alcohol-related liver disease and non-alcoholic fatty liver disease. Seven of the 29 patients with liver disease had mutations in FOXO1, the major transcription factor in insulin signalling. These mutations affected a single hotspot within the gene, impairing the insulin-mediated nuclear export of FOXO1. Notably, six of the seven patients with FOXO1S22W hotspot mutations showed convergent evolution, with variants acquired independently by up to nine distinct hepatocyte clones per patient. CIDEB, which regulates lipid droplet metabolism in hepatocytes17-19, and GPAM, which produces storage triacylglycerol from free fatty acids20,21, also had a significant excess of mutations. We again observed frequent convergent evolution: up to fourteen independent clones per patient with CIDEB mutations and up to seven clones per patient with GPAM mutations. Mutations in metabolism genes were distributed across multiple anatomical segments of the liver, increased clone size and were seen in both alcohol-related liver disease and non-alcoholic fatty liver disease, but rarely in hepatocellular carcinoma. Master regulators of metabolic pathways are a frequent target of convergent somatic mutation in alcohol-related and non-alcoholic fatty liver disease.

The future of liver transplantation.

Over the last 50 years, liver transplantation has evolved into a procedure routinely performed in many countries worldwide. Those able to access this therapy frequently experience a miraculous risk-benefit ratio, particularly if they face the imminently life-threatening disease. Over the decades, the success of liver transplantation, with dramatic improvements in early posttransplant survival, has aggressively driven demand. However, despite the emergence of living donors to augment deceased donors as a source of organs, supply has lagged far behind demand. As a result, rationing has been an unfortunate focus in recent decades. Recent shifts in the epidemiology of liver disease combined with transformative innovations in liver preservation suggest that the underlying premise of organ shortage may erode in the foreseeable future. The focus will sharpen on improving equitable access while mitigating constraints related to workforce training, infrastructure for organ recovery and rehabilitation, and their associated costs. Research efforts in liver preservation will undoubtedly blossom with the aim of optimizing both the timing and conditions of transplantation. Coupled with advances in genetic engineering, regenerative biology, and cellular therapies, the portfolio of innovation, both broad and deep, offers the promise that, in the future, liver transplantation will not only be broadly available to those in need but also represent a highly durable life-saving therapy.

Bouveret Syndrome: A Systematic Review of Endoscopic Therapy and a Novel Predictive Tool to Aid in Management.

BACKGROUND AND GOALS: Bouveret syndrome is characterized by gastroduodenal obstruction caused by an impacted gallstone. Current literature recommends endoscopic therapy as the first line of intervention despite significantly lower success rates compared with surgery. The lack of treatment efficacy studies and the paucity of clinical guidelines contribute to current practices being arbitrary. The aim of this systematic review was to identify factors that predict outcomes of endoscopic therapy. Subsequently, a predictive tool was devised to predict the success of endoscopic therapy and recommendations were proposed to improve current management strategies of impacted gallstones in the upper gastrointestinal tract. METHODS: A systematic search of PubMed, Medline, Cochrane, and Scopus was performed for articles that contained the terms "Bouveret syndrome," "Bouveret's syndrome," "gallstone" AND "gastric obstruction" and "gallstone" AND "duodenal obstruction" that were published between January 1, 1950 to April 15, 2018. Articles were reviewed by 3 reviewers and raw data collated. χ and Kolmogorov-Smirnov tests were used to test associations between predictors and endoscopic outcomes. A logistic regression model was then used to create a predictive tool which was cross validated. RESULTS: Failure of endoscopic therapy is associated with increasing gallstone length (P<0.0001) and impaction in the distal duodenum (P<0.05). Using multiple endoscopic modalities is associated with better success rates (P<0.05). The novel predictive tool predicted success of endoscopic therapy with an area under the receiver operating characteristic score of 0.86 (95% confidence interval: 0.79-0.94). CONCLUSION: In Bouveret syndrome, a selective approach to endoscopic therapy can expedite definitive treatment and improve current management strategies.

Mutational History of a Human Cell Lineage from Somatic to Induced Pluripotent Stem Cells.

The accuracy of replicating the genetic code is fundamental. DNA repair mechanisms protect the fidelity of the genome ensuring a low error rate between generations. This sustains the similarity of individuals whilst providing a repertoire of variants for evolution. The mutation rate in the human genome has recently been measured to be 50-70 de novo single nucleotide variants (SNVs) between generations. During development mutations accumulate in somatic cells so that an organism is a mosaic. However, variation within a tissue and between tissues has not been analysed. By reprogramming somatic cells into induced pluripotent stem cells (iPSCs), their genomes and the associated mutational history are captured. By sequencing the genomes of polyclonal and monoclonal somatic cells and derived iPSCs we have determined the mutation rates and show how the patterns change from a somatic lineage in vivo through to iPSCs. Somatic cells have a mutation rate of 14 SNVs per cell per generation while iPSCs exhibited a ten-fold lower rate. Analyses of mutational signatures suggested that deamination of methylated cytosine may be the major mutagenic source in vivo, whilst oxidative DNA damage becomes dominant in vitro. Our results provide insights for better understanding of mutational processes and lineage relationships between human somatic cells. Furthermore it provides a foundation for interpretation of elevated mutation rates and patterns in cancer.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

Inflammatory Gene Expression in Livers Undergoing Ex Situ Normothermic Perfusion Is Attenuated by Leukocyte Removal From the Perfusate.

BACKGROUND: Ex situ normothermic perfusion (ESNP) is a method to evaluate and potentially recondition organs before transplantation. However, increased expression of inflammatory molecules, including by tissue-resident immune cells, may occur during the perfusion process, potentially negating the beneficial effects of perfusion. METHODS: We used RNA sequencing to assess gene expression in 31 livers undergoing ESNP, including 23 donated after circulatory death (DCD) and 8 donated after brain death. In 7 DCD livers, a leucocyte filter was added to the circuit during perfusion. Biopsies were available for transcriptomic assessment in all cases at the start of perfusion and at varying time points postperfusion. RESULTS: During ESNP in DCD livers, we observed an increase in proinflammatory, profibrinolytic, and prorepair pathway genes. SERPINE1, encoding plasminogen activator inhibitor-1, was among the genes most significantly upregulated during perfusion in DCD livers, potentially promoting fibrin clot persistence in vasculature. We also found increased expression of monocyte and neutrophil recruiting chemokine and proinflammatory cytokine transcripts during ESNP, but several prorepair molecules, including thymic stromal lymphopoietin, were also upregulated. In both DCD and donation after brain death livers, interferon-gamma response genes were enriched, whereas oxidative phosphorylation genes decreased in organs with high perfusate alanine transaminase, a biomarker associated with adverse clinical outcomes. The inclusion of a leukocyte filter in the perfusion circuit mitigated the induction of inflammation/immune pathway genes during perfusion and was associated with enrichment in oxidative phosphorylation genes. CONCLUSIONS: Leukocyte removal during ESNP abrogates transcriptional changes that are associated with unfavorable clinical outcomes, potentially benefiting human livers undergoing ESNP.

Novel stem cell technologies are powerful tools to understand the impact of human factors on Plasmodium falciparum malaria.

Plasmodium falciparum parasites have a complex life cycle, but the most clinically relevant stage of the disease is the invasion of erythrocytes and the proliferation of the parasite in the blood. The influence of human genetic traits on malaria has been known for a long time, however understanding the role of the proteins involved is hampered by the anuclear nature of erythrocytes that makes them inaccessible to genetic tools. Here we overcome this limitation using stem cells to generate erythroid cells with an in-vitro differentiation protocol and assess parasite invasion with an adaptation of flow cytometry to detect parasite hemozoin. We combine this strategy with reprogramming of patient cells to Induced Pluripotent Stem Cells and genome editing to understand the role of key genes and human traits in malaria infection. We show that deletion of basigin ablates invasion while deletion of ATP2B4 has a minor effect and that erythroid cells from reprogrammed patient-derived HbBart α-thalassemia samples poorly support infection. The possibility to obtain patient-secific and genetically modifed erythoid cells offers an unparalleled opportunity to study the role of human genes and polymorphisms in malaria allowing preservation of the genomic background to demonstrate their function and understand their mechanisms.

Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells.

We explored human induced pluripotent stem cells (hiPSCs) derived from different tissues to gain insights into genomic integrity at single-nucleotide resolution. We used genome sequencing data from two large hiPSC repositories involving 696 hiPSCs and daughter subclones. We find ultraviolet light (UV)-related damage in ~72% of skin fibroblast-derived hiPSCs (F-hiPSCs), occasionally resulting in substantial mutagenesis (up to 15 mutations per megabase). We demonstrate remarkable genomic heterogeneity between independent F-hiPSC clones derived during the same round of reprogramming due to oligoclonal fibroblast populations. In contrast, blood-derived hiPSCs (B-hiPSCs) had fewer mutations and no UV damage but a high prevalence of acquired BCOR mutations (26.9% of lines). We reveal strong selection pressure for BCOR mutations in F-hiPSCs and B-hiPSCs and provide evidence that they arise in vitro. Directed differentiation of hiPSCs and RNA sequencing showed that BCOR mutations have functional consequences. Our work strongly suggests that detailed nucleotide-resolution characterization is essential before using hiPSCs.

Abrogation of antibody-mediated allograft rejection by regulatory CD4 T cells with indirect allospecificity.

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.

Investigation of red blood cells from alpha1,3-galactosyltransferase-knockout pigs for human blood transfusion.

BACKGROUND: Pigs are a potential source of red blood cells (RBCs) for transfusion into humans, but the presence of galactose-alpha1,3-galactose (Gal) epitopes on their surface, against which humans have anti-Gal, has been perceived as a major barrier. alpha1,3-Galactosyltransferase gene-knockout pigs, which do not express Gal epitopes on RBCs (Gal-/-), have recently become available. STUDY DESIGN AND METHODS: In vitro, RBCs from Gal-/- pigs were exposed to sera from naïve humans or baboons or from baboons previously sensitized to pig antigens; immunoglobulin binding was measured by flow cytometry, and cytotoxicity, by a hemolytic assay. In vivo, relatively small numbers of Gal-/- RBCs were transfused into two nonsensitized untreated baboons. The survival of pig RBCs was detected by flow cytometry. RESULTS: In vitro, binding of immunoglobulin (Ig) M from naïve human or baboon sera was detected to Gal-/- RBCs but was significantly less than to Gal+/+ RBCs; IgG binding to Gal-/- RBCs was absent or minimal. Sera had minimal cytotoxicity to Gal-/- RBCs compared to Gal+/+ RBCs. Sensitized baboon sera demonstrated much higher IgG binding to Gal-/- RBCs and increased cytotoxicity, but again these were less than to Gal+/+ RBCs. In vivo, the transfusion of relatively small volumes of Gal-/- RBCs was followed by detection of the cells in the baboon's blood for only 5 minutes. CONCLUSION: Pig RBCs are rapidly phagocytosed from the primate circulation by a mechanism not involving anti-Gal.