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Fatty liver disease has grown into a major global health burden, attributed to multi-factors including sedentary lifestyle, obesogenic diet and prevalence of metabolic disorders. The lack of robust experimental models is hampering the research and therapeutic development for fatty liver disease. This study aims to develop an organoid-based 3D culture model to recapitulate key features of fatty liver disease focusing on intracellular lipid accumulation and metabolic dysregulation. We used human liver-derived intrahepatic cholangiocyte organoids and hepatocyte differentiated organoids. These organoids were exposed to lactate, pyruvate, and octanoic acid (LPO) for inducing lipid accumulation and mitochondrial impairment. Lipid accumulation resulted in alternations of gene transcription with major effects on metabolic pathways, including triglyceride and glucose level increase, which is consistent with metabolic changes in fatty liver disease patients. Interestingly, lipid accumulation affected mitochondria as shown by morphological transitions, alternations in expression of mitochondrial encoded genes, and reduction of ATP production. Meanwhile, we found treatment with obeticholic acid and metformin can alleviate fat accumulation in organoids. This study demonstrated that LPO exposure can induce lipid accumulation and associated metabolic dysregulation in human liver-derived organoids. This provides an innovative model for studying fatty liver disease and testing potential therapeutics. Key messages: Lactate, pyruvate, and octanoic acid induce lipid accumulation in liver organoids.Organoids of human compared to mouse origin are more efficient in lipid accumulation.Lipid accumulation dysregulates metabolic pathway and impairs mitochondrial function.Demonstrating a proof-of-concept for testing medications in organoids.

Background & Aims Liver and bile duct diseases often are associated with extensive cell death of cholangiocytes. Necroptosis represents a common mode of programmed cell death in cholangiopathy, however, detailed mechanistic knowledge is limited owing to the lack of appropriate in vitro models. To address this void, we investigated whether human intrahepatic cholangiocyte organoids (ICOs) can recapitulate cholangiopathy-associated necroptosis and whether this model can be used for drug screening. Methods We evaluated the clinical relevance of necroptosis in end-stage liver diseases and liver transplantation by immunohistochemistry. Cholangiopathy-associated programmed cell death was evoked in ICOs derived from healthy donors or patients with primary sclerosing cholangitis or alcoholic liver diseases by the various stimuli. Results The expression of key necroptosis mediators, receptor-interacting protein 3 and phosphorylated mixed lineage kinase domain-like, in cholangiocytes during end-stage liver diseases was confirmed. The phosphorylated mixed lineage kinase domain-like expression was etiology-dependent. Gene expression analysis confirmed that primary cholangiocytes are more prone to necroptosis compared with primary hepatocytes. Both apoptosis and necroptosis could be specifically evoked using tumor necrosis factor α and second mitochondrial-derived activator of caspases mimetic, with or without caspase inhibition in healthy and patient-derived ICOs. Necroptosis also was induced by ethanol metabolites or human bile in ICOs from donors and patients. The organoid cultures further uncovered interdonor variable and species-specific drug responses. Dabrafenib was identified as a potent necroptosis inhibitor and showed a protective effect against ethanol metabolite toxicity. Conclusions Human ICOs recapitulate cholangiopathy-associated necroptosis and represent a useful in vitro platform for the study of biliary cytotoxicity and preclinical drug evaluation., Graphical abstract

Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.

Inflammatory liver disease increases the risk of developing primary liver cancer. The mechanism through which liver disease induces tumorigenesis remains unclear, but is thought to occur via increased mutagenesis. Here, we performed whole-genome sequencing on clonally expanded single liver stem cells cultured as intrahepatic cholangiocyte organoids (ICOs) from patients with alcoholic cirrhosis, non-alcoholic steatohepatitis (NASH), and primary sclerosing cholangitis (PSC). Surprisingly, we find that these precancerous liver disease conditions do not result in a detectable increased accumulation of mutations, nor altered mutation types in individual liver stem cells. This finding contrasts with the mutational load and typical mutational signatures reported for liver tumors, and argues against the hypothesis that liver disease drives tumorigenesis via a direct mechanism of induced mutagenesis. Disease conditions in the liver may thus act through indirect mechanisms to drive the transition from healthy to cancerous cells, such as changes to the microenvironment that favor the outgrowth of precancerous cells., Nguyen et al. used liver organoid cultures to identify all somatic mutations in individual stem cells from patients with common liver diseases that confer risk of cancer. The authors report that, compared to healthy liver, these precancerous liver diseases do not result in a detectable increase of mutations, nor an alteration of mutation types in individual liver stem cells.

Background Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. Methods CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. Results The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell–cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. Conclusions In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

Organoid technology holds great promise for regenerative medicine, but has not yet been applied to humans. With the ground breaking paper in Science (1), by Sampaziotis and colleagues from the University of Cambridge, UK, clinical applications of cholangiocyte organoids come in sight. The authors describe how cholangiocyte organoids can be used in the context of cholangiopathies and show bile duct repair by successfully transplanting these organoids in the human liver. Using single-cell RNA sequencing, they furthermore demonstrate transcriptional diversity in primary human cholangiocytes and that this is mostly lost in organoid culture.

Background & Aims Cancer-associated fibroblasts (CAFs) play a key role in the cancer process, but the research progress is hampered by the paucity of preclinical models that are essential for mechanistic dissection of cancer cell–CAF interactions. Here, we aimed to establish 3-dimensional (3D) organotypic co-cultures of primary liver tumor–derived organoids with CAFs, and to understand their interactions and the response to treatment. Methods Liver tumor organoids and CAFs were cultured from murine and human primary liver tumors. 3D co-culture models of tumor organoids with CAFs and Transwell culture systems were established in vitro. A xenograft model was used to investigate the cell–cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma cohort and an online liver cancer database indicated the clinical relevance of CAFs. Results To functionally investigate the interactions of liver cancer cells with CAFs, we successfully established murine and human 3D co-culture models of liver tumor organoids with CAFs. CAFs promoted tumor organoid growth in co-culture with direct cell–cell contact and in a Transwell system via paracrine signaling. Vice versa, cancer cells secrete paracrine factors regulating CAF physiology. Co-transplantation of CAFs with liver tumor organoids of mouse or human origin promoted tumor growth in xenograft models. Moreover, tumor organoids conferred resistance to clinically used anticancer drugs including sorafenib, regorafenib, and 5-fluorouracil in the presence of CAFs, or the conditioned medium of CAFs. Conclusions We successfully established murine and human 3D co-culture models and have shown robust effects of CAFs in liver cancer nurturing and treatment resistance., Graphical abstract

Background: In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR)-dependent bicarbonate secretion precipitates the accumulation of viscous mucus in the lumen of respiratory and gastrointestinal epithelial tissues. We investigated whether the combination of elexacaftor (ELX), ivacaftor (IVA) and tezacaftor (TEZ), apart from its well-documented effect on chloride transport, also restores Phe508del-CFTR-mediated bicarbonate transport. Methods: Epithelial monolayers were cultured from intestinal and biliary (cholangiocyte) organoids of homozygous Phe508del-CFTR patients and controls. Transcriptome sequencing was performed, and bicarbonate and chloride transport were assessed in the presence or absence of ELX/IVA/TEZ, using the intestinal current measurement technique. Results: ELX/IVA/TEZ markedly enhanced bicarbonate and chloride transport across intestinal epithelium. In biliary epithelium, it failed to enhance CFTR-mediated bicarbonate transport but effectively rescued CFTR-mediated chloride transport, known to be requisite for bicarbonate secretion through the chloride-bicarbonate exchanger AE2 (SLC4A2), which was highly expressed by cholangiocytes. Biliary but not intestinal epithelial cells expressed an alternative anion channel, anoctamin-1/TMEM16A (ANO1), and secreted bicarbonate and chloride upon purinergic receptor stimulation. Conclusions: ELX/IVA/TEZ has the potential to restore both chloride and bicarbonate secretion across CF intestinal and biliary epithelia and may counter luminal hyper-acidification in these tissues.

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding β‐catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy., Prime editing uses Cas9 nickase fused to a reverse transcriptase to edit genetic information. Here, the authors prime edit primary adult stem cells in 3D organoid cultures to show functional correction of pathogenic mutations without genome-wide off-target effects.

Background and Aims: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)–positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Approach and Results: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. Conclusions: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are thought to be the main drivers for disease progression and treatment resistance across various cancer types. Identifying and targeting these rare cancer cells, however, remains challenging with respect to therapeutic benefit. Here, we report the enrichment of LGR5 expressing cells, a well-recognized stem cell marker, in mouse liver tumors, and the upregulation of LGR5 expression in human hepatocellular carcinoma. Isolated LGR5 expressing cells from mouse liver tumors are superior in initiating organoids and forming tumors upon engraftment, featuring candidate TICs. These cells are resistant to conventional treatment including sorafenib and 5-FU. Importantly, LGR5 lineage ablation significantly inhibits organoid initiation and tumor growth. The combination of LGR5 ablation with 5-FU, but not sorafenib, further augments the therapeutic efficacy in vivo. Thus, we have identified the LGR5+ compartment as an important TIC population, representing a viable therapeutic target for combating liver cancer., Cancer stem cells (CSCs) are thought to be the main drivers for disease progression and treatment resistance in liver cancer. This study identifies the LGR5+ compartment as an important CSC population, representing a viable therapeutic target for combating liver cancer.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modelling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease) and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process. This article is protected by copyright. All rights reserved.

For liver transplantations, human leukocyte antigen (HLA) matching is not routinely performed because observed effects have been inconsistent. Nevertheless, long-term liver transplantation outcomes remain suboptimal. The availability of a more precise HLA-matching algorithm, Predicted Indirectly Recognizable HLA Epitopes II (PIRCHE-II), now enables robust assessment of the association between HLA matching and liver transplantation outcomes. We performed a single-center retrospective cohort study of 736 liver transplantation patients. Associations between PIRCHE-II and HLAMatchmaker scores and mortality, graft loss, acute and chronic rejection, ischemic cholangiopathy, and disease recurrence were evaluated with Cox proportional hazards models. Associations between PIRCHE-II with 1-year, 2-year, and 5-year outcomes and severity of acute rejection were assessed with logistic and linear regression analyses, respectively. Subgroup analyses were performed for autoimmune and nonautoimmune indications, and patients aged 30 years and younger, and older than 30 years. PIRCHE-II and HLAMatchmaker scores were not associated with any of the outcomes. However, patients who received transplants for autoimmune disease showed more acute rejection and graft loss, and these risks negatively associated with age. Rhesus mismatch more than doubled the risk of disease recurrence. Moreover, PIRCHE-II was inversely associated with graft loss in the subgroup of patients aged 30 years and younger with autoimmune indications. The absence of associations between PIRCHE-II and HLAMatchmaker scores and the studied outcomes refutes the need for HLA matching for liver (stem cell) transplantations for nonautoimmune disease. For autoimmune disease, the activated immune system seems to increase risks of acute rejection and graft loss. Our results may suggest the benefits of transplantations with rhesus matched but PIRCHE-II mismatched donor livers.

Hepatotropic viruses naturally have narrow host and tissue tropisms, challenging the development of robust experimental models. The advent of organoid technology provides a unique opportunity for moving the field forward. Here, we demonstrate that three-dimensional cultured organoids from fetal and adult human liver with cholangiocyte or hepatocyte phenotype support hepatitis E virus (HEV) replication. Inoculation with infectious HEV particles demonstrates that human liver–derived organoids support the full life cycle of HEV infection. By directing organoids toward polarized monolayers in a transwell system, we observed predominantly apical secretion of HEV particles. Genome-wide transcriptomic and tRNAome analyses revealed robust host responses triggered by viral replication. Drug screening in organoids identified brequinar and homoharringtonine as potent HEV inhibitors, which are also effective against the ribavirin resistance variant harboring G1634R mutation. Thus, successful recapitulation of HEV infection in liver-derived organoids shall facilitate the study of virus-host interactions and development of antiviral therapies., Description, Human liver–derived organoids support HEV infection and enable the study of virus-host interaction and antiviral drug development.

In three-dimensional light microscopy, the heterogeneity of the optical density in a specimen ultimately limits the achievable penetration depth and hence the three-dimensional resolution. The most direct approach to reduce aberrations, improve the contrast and achieve an optimal resolution is to minimise the impact of changes of the refractive index along an optical path. Many implementations of light sheet fluorescence microscopy operate with a large chamber filled with an aqueous immersion medium and a further inner container with the specimen embedded in a possibly entirely different non-aqueous medium. In order to minimise the impact of the latter on the optical quality of the images, we use multi-facetted cuvettes fabricated from vacuum-formed ultra-thin fluorocarbon (FEP) foils. The ultra-thin FEP-foil cuvettes have a wall thickness of about 10–12 µm. They are impermeable to liquids, but not to gases, inert, durable, mechanically stable and flexible. Importantly, the usually fragile specimen can remain in the same cuvette from seeding to fixation, clearing and observation, without the need to remove or remount it during any of these steps. We confirm the improved imaging performance of ultra-thin FEP-foil cuvettes with excellent quality images of whole organs such us mouse oocytes, of thick tissue sections from mouse brain and kidney as well as of dense pancreas and liver organoid clusters. Our ultra-thin FEP-foil cuvettes outperform many other sample-mounting techniques in terms of a full separation of the specimen from the immersion medium, compatibility with aqueous and organic clearing media, quick specimen mounting without hydrogel embedding and their applicability for multiple-view imaging and automated image segmentation. Additionally, we show that ultra-thin FEP foil cuvettes are suitable for seeding and growing organoids over a time period of at least ten days. The new cuvettes allow the fixation and staining of specimens inside the holder, preserving the delicate morphology of e.g. fragile, mono-layered three-dimensional organoids.

Cell death is a natural process for the turnover of aged cells, but it can also arise as a result of pathological conditions. Cell death is recognized as a key feature in both acute and chronic hepatobiliary diseases caused by drug, alcohol, and fat uptake; by viral infection; or after surgical intervention. In the case of chronic disease, cell death can lead to (chronic) secondary inflammation, cirrhosis, and the progression to liver cancer. In liver transplantation, graft preservation and ischemia/reperfusion injury are associated with acute cell death. In both cases, so-called programmed cell death modalities are involved. Several distinct types of programmed cell death have been described of which apoptosis and necroptosis are the most well known. Parenchymal liver cells, including hepatocytes and cholangiocytes, are susceptible to both apoptosis and necroptosis, which are triggered by distinct signal transduction pathways. Apoptosis is dependent on a proteolytic cascade of caspase enzymes, whereas necroptosis induction is caspase-independent. Moreover, different from the "silent" apoptotic cell death, necroptosis can cause a secondary inflammatory cascade, so-called necroinflammation, triggered by the release of various damage-associated molecular patterns (DAMPs). These DAMPs activate the innate immune system, leading to both local and systemic inflammatory responses, which can even cause remote organ failure. Therapeutic targeting of necroptosis by pharmacological inhibitors, such as necrostatin-1, shows variable effects in different disease models.

Mitochondria are essential organelles for cellular energy and metabolism. Like with any organ, the liver highly depends on the function of these cellular powerhouses. Hepatotoxic insults often lead to an impairment of mitochondrial activity and an increase in oxidative stress, thereby compromising the metabolic and synthetic functions. Mitochondria play a critical role in ATP synthesis and the production or scavenging of free radicals. Mitochondria orchestrate many cellular signalling pathways involved in the regulation of cell death, metabolism, cell division and progenitor cell differentiation. Mitochondrial dysfunction and oxidative stress are closely associated with ischemia-reperfusion injury during organ transplantation and with different liver diseases, including cholestasis, steatosis, viral hepatitis, and drug-induced liver injury. In order to develop novel mitochondria-targeting therapies or interventions, a better understanding of mitochondrial dysfunction and oxidative stress in hepatic pathogenesis is very much needed. Therapies targeting mitochondria impairment and oxidative imbalance in liver diseases have been extensively studied in preclinical and clinical research. In this review, we provide an overview of how oxidative stress and mitochondrial dysfunction affect liver diseases and liver transplantation. Furthermore, we summarize recent developments of antioxidant and mitochondria-targeted interventions.

Diseases of the bile duct (cholangiopathies) remain a common indication for liver transplantation, while little progress has been made over the last decade in understanding the underlying pathophysiology. This is largely due to lack of properin vitromodel systems to study cholangiopathies. Recently, a culture method has been developed that allows for expansion of human bile duct epithelial cells grown as extrahepatic cholangiocyte organoids (ncECOs) in non-canonical Wnt-stimulating conditions. These ncECOs closely resemble cholangiocytes in culture and have shown to efficiently repopulate collagen scaffolds that could act as functional biliary tissue in mice. Thus far, initiation of ncECOs required tissue samples, thereby limiting broad patient-specific applications. Here, we report that bile fluid, which can be less invasively obtained and with low risk for the patients, is an alternative source for culturing ncECOs. Further characterization showed that bile-derived cholangiocyte organoids (ncBCOs) are highly similar to ncECOs obtained from bile duct tissue biopsies. Compared to the previously reported bile-cholangiocyte organoids cultured in canonical Wnt-stimulation conditions, ncBCOs have superior function of cholangiocyte ion channels and are able to respond to secretin and somatostatin. In conclusion, bile is a new, less invasive, source for patient-derived cholangiocyte organoids and makes their regenerative medicine applications more safe and feasible.

Cholangiocytes express cystic fibrosis transmembrane conductance regulator (CFTR), which is involved in bicarbonate secretion for the protection against bile toxicity. During liver transplantation, prolonged hypoxia of the graft is associated with cholangiocyte loss and biliary complications. Hypoxia is known to diminish CFTR activity in the intestine, but whether it affects CFTR activity in cholangiocytes remains unknown. Thus, the aim of this study is to investigate the effect of hypoxia on CFTR activity in intrahepatic cholangiocyte organoids (ICOs) and test drug interventions to restore bicarbonate secretion. Fifteen different human ICOs were cultured as monolayers and ion channel [CFTR and anoctamin-1 (ANO1)] activity was determined using an Ussing chamber assay with or without AMP kinase (AMPK) inhibitor under hypoxic and oxygenated conditions. Bile toxicity was tested by apical exposure of cells to fresh human bile. Overall gene expression analysis showed a high similarity between ICOs and primary cholangiocytes. Under oxygenated conditions, both CFTR and ANO1 channels were responsible for forskolin and uridine- 50-triphosphate (UTP) UTP-activated anion secretion. Forskolin stimulation in the absence of intracellular chloride showed ion transport, indicating that bicarbonate could be secreted by CFTR. During hypoxia, CFTR activity significantly decreased (P = 0.01). Switching from oxygen to hypoxia during CFTR measurements reduced CFTR activity (P = 0.03). Consequently, cell death increased when ICO monolayers were exposed to bile during hypoxia compared with oxygen (P = 0.04). Importantly, addition of AMPK inhibitor restored CFTR-mediated anion secretion during hypoxia. ICOs provide an excellent model to study cholangiocyte anion channels and drug-related interventions. Here, we demonstrate that hypoxia affects cholangiocyte ion secretion, leaving cholangiocytes vulnerable to bile toxicity. The mechanistic insights from this model maybe relevant for hypoxia-related biliary injury during liver transplantation.

There is substantial evidence indicating the possible involvement of rotavirus in biliary atresia (BA) development, at least in a subset of patients, but concrete proof remains lacking. In a mouse model, it has been well demonstrated that rotavirus can infect the biliary epithelium to cause biliary inflammation and obstruction, representing the pathogenesis of BA in humans. By using recently developed organoids technology, we now have demonstrated that human biliary organoids are susceptible to rotavirus infection, and this provokes active virus-host interactions and causes severe cytopathogenesis. Thus, our model recapitulates some essential aspects of BA development. Furthermore, we have demonstrated that antiviral drugs and neutralizing antibodies are capable of counteracting the infection and BA-like morphological changes, suggesting their potential for mitigating BA in patients., Biliary atresia (BA) is a neonatal liver disease characterized by progressive fibroinflammatory obliteration of both intrahepatic and extrahepatic bile ducts. The etiologies of BA remain largely unknown, but rotavirus infection has been implicated at least for a subset of patients, and this causal relation has been well demonstrated in mouse models. In this study, we aim to further consolidate this evidence in human biliary organoids. We obtained seven batches of human biliary organoids cultured from fetal liver, adult liver, and bile duct tissues. We found that these organoids are highly susceptible and support the full life cycle of rotavirus infection in three-dimensional culture. The robust infection triggers active virus-host interactions, including interferon-based host defense mechanisms and injury responses. We have observed direct cytopathogenesis in organoids upon rotavirus infection, which may partially recapitulate the development of BA. Importantly, we have demonstrated the efficacy of mycophenolic acid and interferon alpha but not ribavirin in inhibiting rotavirus in biliary organoids. Furthermore, neutralizing antibody targeting rotavirus VP7 protein effectively inhibits infection in organoids. Thus, we have substantiated the causal evidence of rotavirus inducing BA in humans and provided potential strategies to combat the disease.

Organoids are morphologically heterogeneous three-dimensional cell culture systems. To understand the cell organisation principles of their morphogenesis, we imaged hundreds of pancreas and liver organoids in parallel using light sheet and bright field microscopy for up to seven days. We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation and degeneration, and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright field macroscale analyses of entire cultures. Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying core regulatory principles of organoid morphogenesis.Graphical AbstractCreated with BioRender.com.

Biliary disorders can lead to life‐threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue‐specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte‐markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs., Willemse and coworkers successfully decellularized human extrahepatic bile duct tissue. Organoids from three different hepatobiliary sources (intrahepatic bile ducts, extrahepatic bile ducts, and bile) were tested for their repopulation efficiency on the decellularized ductal matrix. Bile‐ and extrahepatic bile duct‐derived organoids were capable of repopulating the ductal matrix, expressed cholangiocyte‐specific markers, and restored barrier function.

Introduction The co-occurrence of inflammatory bowel disease (IBD) in up to 80% of patients with primary sclerosing cholangitis (PSC) suggests a relation between the gut and the liver in patients with both PSC and IBD. One hypothesis suggests that aberrantly expressed homing molecules in the liver drive infiltration of gut-homing memory T-cells that are originally primed in intestinal environment. One of the main findings supporting this hypothesis is the expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in PSC livers. Expression of homing molecules in early PSC remains unclear. The aim of this study was to investigate expression patterns of homing chemokines and adhesion molecules in PSC-IBD colons and livers, and to study whether changes are already present in early stages of PSC. Methods Needle biopsies from livers of 20 PSC patients with short-term PSC (PSC-IBDST) as well as explant liver biopsies of 8 patients with long-term PSC (PSC-IBDLT) were collected (median disease duration 0 and 22 years, respectively). Only patients with concomitant IBD were included (89% ulcerative colitis and 11% Crohn’s disease). Expression and distribution of MAdCAM-1, VAP-1, integrin β7, CCL25, CCL28, CXCL12, αE (CD103) and E-cadherin were assessed in both liver and colon tissue. Liver tissue collected from obstructive cholangitis in resection specimens for Klatskin tumors or resection specimens from hepatic metastasis, liver tissue of patients with hepatitis C virus (HCV) and of patients with primary biliary cholangitis (PBC) served as controls. Results MAdCAM-1 expression in livers of PSC-IBDLT patients was increased compared to controls. The proportion of CD3+ T-cells expressing integrin β7 did not differ between PSC-IBDST and control groups, but was higher in liver tissue of PSC-IBDLT patients. There was no difference in αE+ T-cells between PSC-IBDLT and control groups. The chemokine CCL28 was highly expressed in biliary epithelial cells. This intense staining pattern was more pronounced in PSC-IBDST, but overall did not significantly differ from controls. Conclusions We confirm that aberrant gut lymphocyte homing to the liver exists in PSC, linking gut and liver disease pathology in PSC-IBD. Our data suggests that this phenomenon increases over time in later stages of the disease, worsening ongoing inflammation., Highlights • MAdCAM-1 is aberrantly expressed in PSC-IBD liver compared to control liver. • The proportion of beta7 positive T-cells is increased in long-term PSC livers. • Biliary epithelial cells express CCL28, already in short-term PSC. • Expression patterns of inflammatory cytokines in PSC-IBD colon are not distinct from IBD.

The well‐established 3D organoid culture method enabled efficient expansion of cholangiocyte‐like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal‐invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD‐ and EHBD‐tissue. We demonstrate successful organoid‐initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional‐specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP‐derived BCOs and gallbladder‐derived BCOs expressing gallbladder‐specific genes. Moreover, BCOs have limited hepatocyte‐fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid‐initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte‐like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid‐initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine., Lay summary: Cholangiocyte organoids provide a powerful tool for characterizing bile duct epithelium and expanding cholangiocytes for tissue engineering purposes. However, organoid initiation typically requires invasive tissue‐biopsies obtained via surgical procedures associated with complications. To overcome this limitation, organoid cultures were initiated from minimal invasive bile‐samples obtained during routine clinical procedures. Characterization revealed that these bile‐cholangiocyte organoids (BCOs) originate from extrahepatic biliary tissue and are capable of repopulating human extrahepatic bile duct scaffolds. Upon repopulation of scaffolds in vitro, the cells obtain a transcriptomic profile more closely resembling primary cholangiocytes. By using bile as novel source for extrahepatic cholangiocyte organoids, bile duct tissue engineering as well as personalized disease modelling become feasible.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.

Due to the complex function and structure of the liver, resourceful solutions for treating end-stage liver disease are required. Currently, liver transplantation is the only curative therapeutic option. However, due to a worldwide donor shortage, researchers have been looking in other fields for alternative sources of transplantable liver tissue. Recent advances in our understanding of liver physiology, stem cell and matrix biology, have accelerated tissue engineering research. Most notable is the discovery of a culture system to grow liver-like organoids from human hepatic stem cells. The extensive expansion capacity of these stem cells has contributed greatly to the availability of hepatocyte-like cells for tissue engineering. In addition, new techniques are explored to obtain biological liver scaffolds from full size donor organs. This review summarizes these state-of-art techniques which may lay the groundwork towards re-creating transplantable tissue from autologous or allogenic stem cells in the coming decade.

Background & Aims Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. Methods We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. Results We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5 + cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. Conclusions Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.

Excessive alcohol consumption increases the risk of developing liver cancer, but the mechanism through which alcohol drives carcinogenesis is as yet unknown. Here, we determined the mutational consequences of chronic alcohol use on the genome of human liver stem cells prior to cancer development. No change in base substitution rate or spectrum could be detected. Analysis of the trunk mutations in an alcohol-related liver tumor by multi-site whole-genome sequencing confirms the absence of specific alcohol-induced mutational signatures driving the development of liver cancer. However, we did identify an enrichment of nonsynonymous base substitutions in cancer genes in stem cells of the cirrhotic livers, such as recurrent nonsense mutations in PTPRK that disturb Epidermal Growth Factor (EGF)-signaling. Our results thus suggest that chronic alcohol use does not contribute to carcinogenesis through altered mutagenicity, but instead induces microenvironment changes which provide a ‘fertile ground’ for selection of cells with oncogenic mutations.

Human whole-liver perfusion-decellularization is an emerging technique for producing bio-scaffolds for tissue engineering purposes. The native liver extracellular matrix (ECM) provides a superior microenvironment for hepatic cells in terms of adhesion, survival and function. However, current decellularization protocols show a high degree of variation in duration. More robust and effective protocols are required, before human decellularized liver ECM can be considered for tissue engineering applications. The aim of this study is to apply pressure-controlled perfusion and test the efficacy of two different detergents in porcine and human livers. To test this, porcine livers were decellularized using two different protocols; a triton-x-100 (Tx100)-only protocol (N = 3) and a protocol in which Tx100 was combined with SDS (N = 3) while maintaining constant pressure of 120 mm Hg. Human livers (N = 3) with different characteristics (age, weight and fat content) discarded for transplantation were decellularized using an adapted version of the Tx-100-only protocol. Decellularization efficacy was determined by histology and analysis of DNA and RNA content. Furthermore, the preservation of ECM components was assessed. After completing the perfusion cycles with detergents the porcine livers from both protocols were completely white and transparent in color. After additional washing steps with water and DNase, the livers were completely decellularized, as no DNA or cell remnants could be detected. The Tx100-only protocol retained 1.5 times more collagen and 2.5 times more sGAG than the livers decellularized with Tx100 + SDS. The Tx100-only protocol was subsequently adapted for decellularizing whole-organ human livers. The human livers decellularized with pressure-controlled perfusion became off-white in color and semi-transparent within 20 h. Livers decellularized without pressure-controlled perfusion took 64–96 h to completely decellularize, but did not become white or transparent. The addition of pressure-controlled flow did remove all cells and double stranded DNA, but did not damage the ultra-structure of the ECM as was analyzed by histology and scanning electron microscopy. In addition, collagens and sGAG were maintained with the decellularized ECM. In conclusion, we established effective, robust and fast decellularization protocols for both porcine and human livers. With this protocol the duration of decellularization for whole-organ human livers has been shortened considerably. The increased pressure and flow did not damage the ECM, as major ECM components remained intact.