Biofabricated scaffolds facilitate bona fide cellular interactions, cell type specification, and the formation of three-dimensional tissue architecture from human pluripotent stem cells (hPSCs). However, xenogenic biomaterials are poorly defined, and synthetic biomaterials remain underdeveloped and understudied, hindering regulatory approval for clinical use and preventing the translation of lab-grown therapies. Here, we describe a protein screen-based hydrogel system biofabricated from physiologically relevant human components. We show that ‘Alphagel’, a base hydrogel comprising human embryonic matrices, supports the trilineage differentiation of hPSCs into neural, cardiac, and liver tissue. Alphagel is also shown to be biocompatible and biodegradable in vivo . Further, upon adding select proteins from maturing human foetal liver to Alphagel, we show that the resulting hydrogel (termed ‘Hepatogel’) enhances the differentiation of hPSC-derived hepatocytes (H-iHeps) compared with Matrigel. Importantly, when injected into mice livers, Hepatogel significantly improves the retention of H-iHeps compared to standard aqueous cell injections. Altogether, our results provide proof of concept that customisable, organ-specific hydrogel systems are a valuable tool for developing clinically translatable therapies in regenerative medicine and tissue engineering.

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.

Human pluripotent stem cell (hPSC)-based therapies offer promise but pose potential risks due to culture-acquired genetic variants, some of which have been linked with cancer. An international workshop addressed these concerns, highlighting the need for improved strategies to stratify variants and chart a path toward definitive guidelines in hPSC-based therapy.

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.

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.

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.

Liver disease is on the rise, which has created the urgent need for new treatments. An attractive therapeutic approach is the understanding and manipulation of the regenerative pathways of the liver. The regenerative capability of the liver is dependent on the nature of the injury. It is known that mild injury and hepatectomy induce hepatocyte proliferation driven regeneration. However, hepatocyte proliferation is impaired during chronic injury and secondary mechanisms of regeneration might exist. Indeed, studies in animal models have revealed several regenerative processes, which might take place in chronic disease: 1) liver stem cells activation, 2) dedifferentiation/redifferentiation of cholangiocytes/hepatocytes, 3) transdifferentiation between cholangiocytes and hepatocytes. There is little knowledge of the above processes in human and the current knowledge is mainly derived from histopathology analyses. Here we aim to define the mechanisms behind epithelial plasticity in the diseased liver. To understand the liver response to chronic disease, we have collected liver biopsies from just under 50 patients across the spectrum of non-alcoholic fatty liver disease and we performed state of the art 3D imaging and single nuclei RNA sequencing (snRNA). In depth, computational analysis has allowed us to dissect the cellular composition of the liver during the course of chronic liver disease. We further used liver organoids as an in vitro model to validate our snRNAseq findings. The analysis has revealed that disease progression is accompanied by tissue remodelling, loss of zonation and extensive ductular reaction. Further analyses, captured both at the transcriptomic and protein level the presence of cells sharing characteristics from cholangiocytes and hepatocytes, termed biphenotypic cells. Gene enrichment analysis revealed signalling pathways likely involved in their generation. In vitro modelling using NAFLD human derived liver organoids validated these molecular pathways and their importance in the generation of bi-phenotypic cells. In conclusion, we use snRNAseq analysis, to demonstrate the presence of a molecular pathway involved in liver epithelial cell plasticity during chronic liver disease. This study paves the way for the development of new therapies promoting tissue repair to improve organ function.

During a kidney transplant, a plastic tube (stent) is placed in the ureter, connecting the new kidney to the bladder, in order to keep the new join open during the initial phase of transplantation. The stent is then removed after a few weeks via a camera procedure (cystoscopy), as it is no longer needed. The present study compared performing this in the operating theatre or in clinic for transplanted patients using a new single-use type of camera with an integrated grasper system. The results have shown that it is safe and cost-effective to do this in clinic, despite patients being susceptible to infection after transplantation.

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.

Abstract Background Inferior vena cava stenosis (IVCS) is a rare complication of liver transplantation with a reported incidence rate of 3%. Limited clinical consensus exists on the management of IVCS. We report the management and outcomes of patients with IVCS at our transplant centre. Methods Relevant data were collected from adult patients who underwent liver transplantation at our centre between October 2014 and August 2020. These included demographics, investigation and management details with regards to IVCS. Values presented as % of total and median with interquartile range (IQR). Results A total of 636 liver transplants were performed during the study period, of which 48 (7.6%) patients were investigated for possible IVCS. Of those, 14 (2.2% of total) were found to have IVCS, 85.7% (n = 12) were female. Only 2/14 were re-transplants and pre-transplant portal vein thrombus was present in 3 cases (21.4%). 10 livers (71.4%) were DBD donors. Normothermic machine perfusion was used in 4/14 patients. All 14 recipients found to have IVCS had had an implantation using a modified piggyback cavocavostomy technique. The IVCS was identified at a median of 25.5 days (19.7-30.8 days) following transplantation within the suprahepatic IVC in 92.9% (n = 13). Hemi-azygos collateralisation was seen in 4 cases (28.6%). 8 of the 14 recipients underwent intervention for IVCS, 6 patients were managed with balloon venoplasty, 1 patient required an IVC stent and 1 was managed surgically. Six of the recipients with IVCS died, 4 of whom had an intervention for their stenosis and 3 of these were within 90 days of their transplant. Pressures measured at the anastomotic stricture were higher in those who succumbed (median of 21 Vs 12.5 mmHg; p=.017). Conclusions At our centre, cava-replacement technique was not associated with IVCS. Patients with more significant strictures (as evidenced by higher pressures at the anastomotic stenosis) may have an increased mortality risk.

The progression of chronic liver disease to hepatocellular carcinoma is caused by the acquisition of somatic mutations that affect 20-30 cancer genes(1-8). Burdens of somatic mutations are higher and clonal expansions larger in chronic liver disease(9-13) than in normal liver(13-16), which enables positive selection to shape the genomic landscape(9-13). 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 FOXO1(S22W) 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 hepatocytes(17-19), and GPAM, which produces storage triacylglycerol from free fatty acids(20,21), 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.

ABSTRACT Plasmodium falciparum interacts with several human cell types during their complex life cycle, including erythrocytes and hepatocytes. The enuclated nature of erythrocytes makes them inaccessible to genetic tools, which in turn makes studying erythrocyte proteins involved in malaria invasion and development particularly difficult. Here we overcome this limitation using stem cell technology to develop a universal differentiation protocol for in vitro derivation of erythrocytes from a variety of stem cell lines of diverse origin. This allows manipulation of erythrocytic genes and examination of their impact on the parasite by flow cytometric detection of parasite haemozoin. Deletion of Basigin, the essential receptor for P. falciparum , abrogates invasion, while other less studied proteins such as ATP2B4 have a minor effect. Reprogramming of induced pluripotent stem cells from α-thalassemia primary samples shows reduced infection levels, demonstrating this approach is useful for understanding the effect of natural human polymorphisms on the disease.

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.

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. χ2 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.

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.

Human iPS cells have been generated using a diverse range of tissues from a variety of donors using different reprogramming vectors. However, these cell lines are heterogeneous, which presents a limitation for their use in disease modeling and personalized medicine. To explore the basis of this heterogeneity we generated 25 iPS cell lines under normalised conditions from the same set of somatic tissues across a number of donors. RNA-seq data sets from each cell line were compared to identify the majority contributors to transcriptional heterogeneity. We found that genetic differences between individual donors were the major cause of transcriptional variation between lines. In contrast, residual signatures from the somatic cell of origin, so called epigenetic memory, contributed relatively little to transcriptional variation. Thus, underlying genetic background variation is responsible for most heterogeneity between human iPS cell lines. We conclude that epigenetic effects in hIPSCs are minimal, and that hIPSCs are a stable, robust and powerful platform for large-scale studies of the function of genetic differences between individuals. Our data also suggest that future studies using hIPSCs as a model system should focus most effort on collection of large numbers of donors, rather than generating large numbers of lines from the same donor.