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Introduction During chronic liver disease the liver can be regenerated from a population of endogenous hepatic progenitor cells (HPCs). These cells are known to be bipotent and can regenerate both hepatocytes and cholangiocytes, however the mechanisms by which these lineages are delineated from HPCs is currently unknown. Here we describe how the Notch and Wnt signalling pathways are differentially regulated and interact via NUMB to define cholangiocytes and hepatocytes from HPCs. Results We have found that during biliary regeneration in primary sclerosing cholangitis and primary biliary cirrhosis, as well as mouse models of such, the Notch pathway is activated as demonstrated through nuclear Notch-1 in HPCs. This Notch mechanism is mediated through interaction of Jagged-1 supplied by a transient myofibroblast niche in mouse, or autonomous cell signalling in established human disease. In vitro when murine HPCs are co-cultured with myofibroblasts y-secretase inhibition significantly reduced expression of the Notch effectors HeyL (p<0.001 3.133±0.32 vs 0.98±0.09) and Hey1 (p<0.001 1.67±0.34 vs 0.08±0.01); associated with this reduction we found a significant reduction in expression of biliary genes HNF6 (p<0.001 77.94±12.52 vs 14.97±4.57) and GGT (p<0.01 6082±757.70 vs 3169±449.0). In vivo administration of DAPT during murine biliary regeneration demonstrates a significant reduction of cholangiocyte numbers (p<0.05 957.7±114.2 vs 1787±266) and also a reduction in pro-biliary genes including HNF6 and HNF1β. NUMB, a negative regulator of Notch signalling is lost during murine biliary regeneration (p<0.01; 1.19±0.07 vs 0.10±0.02) as well as during PSC/PBC (p<0.01; 8748±3090 vs 1814±277). During regeneration of hepatocytes from HPCs in HCV and in murine models NUMB levels remain high and Notch signalling is suppressed. During murine hepatocyte regeneration NUMB is maintained via macrophage derived Wnt. In vitro activation of the Wnt pathway in HPCs resulted in a twofold induction of NUMB and 35-fold induction of HNF4, without significantly affecting HNF1β. We ablated macrophages using liposomal clodronate this resulted in a conversion of hepatocyte HPCs into biliary HPCs which formed luminal structures with membranous b-catenin, lost expression of Wnt pathway target Axin2, NUMB and HNF4 as well as inducing expression of Hes-1, HNF1β and HNF6. Conclusion During regeneration of the adult liver, Notch signalling is required for biliary specification however is actively sequestered via NUMB during regeneration of hepatocytes. This hepatocyte phenotype is induced through signalling via the Wnt pathway, removal of which results in a biliary phenotype during hepatocyte regeneration. We also describe how this mechanism is conserved from mouse models to human disease, where during biliary diseases such as PSC and PBC the Notch pathway is activated, however is actively restricted during regeneration of in hepatocytes during HCV. [ABSTRACT FROM PUBLISHER]

Introduction Autologous bone marrow cell (BMC) therapy for liver disease has shown increased liver regeneration in animal studies and in phase 1 clinical studies. As yet no clear mechanism accounts for these observations. BMCs do not directly form into hepatocytes but can improve liver regeneration and function. In chronic liver disease hepatic progenitor cells (HPCs) are a potential source of parenchymal regeneration. Whilst the transplantation of human HPCs is not currently a practical therapeutic option, manipulating endogenous HPCs in vivo represents a potential approach. HPCs exist in a specialised niche of leukocytes and mesenchymal cells, which secrete cytokines that are capable of regulating HPC behaviour. We therefore hypothesised that infusion of BMC into the liver may induce HPC expansion via paracrine signalling. Aim To investigate the effect of autologous BMC infusion upon regeneration by hepatic progenitor cells. Method 107 syngenic BMCs were infused into healthy mice by tail vein, we examined intrahepatic donor cell engraftment, cytokine expression, liver function tests, and HPC activation. Cell tracking utilised either GFP+ or male cells delivered into female wild type mice. Whole liver and specific cell fractions were analysed by immunocytochemistry, in-situ hybridisation, FACS, and qRT-PCR. Results Following BMC transfer, a progressive and sustained expansion of HPCs was observed (mean±SEM 41.9±2.1 cells per field vs PBS control 23.5±4.1 at 21 days post infusion, p=0.003) with associated increase in liver/body weight ratio (BMC 0.0513±0.001 vs PBS control 0.0470±0.001, p=0.022). BMCs engrafted the liver adjacent to HPCs for up to 3 weeks and were mostly F4/80+ macrophages (81%). Transfer of F4/80+ macrophages alone into healthy mice recapitulated the HPC expansion. Cytokine gene expression analysis revealed one soluble signal in particular, TWEAK, increased in the recipient's liver following donor BMC engraftment (5.20-fold induction vs control at Day 3). Extracted donor derived BMCs expressed TWEAK, as do macrophages. TWEAK is known to be a direct mitogen to HPCs via the Fn14 receptor. When Fn14-/- mice were used as recipients to wild type BMC infusions the expansion of HPCs was entirely lost (mean±SEM 27.2±1.4 vs negative PBS control 30.6±2.4, p=0.128 vs positive wild-type control 42.7±2.3, p=0.0002), demonstrating that the HPC expansion is dependent on TWEAK-Fn14 paracrine signalling between BMCs and HPCs. Conclusion These data describe a hitherto unknown mechanism by which infused autologous macrophages signal in a paracrine manner to HPCs via TWEAK. These observations suggest potential for the development of novel therapies to promote human liver regeneration. [ABSTRACT FROM PUBLISHER]

Background & Aims: Biliary complications are a major cause of morbidity and mortality in liver transplantation. Up to 25% of patients that develop biliary complications require additional surgical procedures, re-transplantation or die in the absence of a suitable regraft. Here, we investigate the role of the primary cilium, a highly specialised sensory organelle, in biliary injury leading to post-transplant biliary complications., Methods: Human biopsies were used to study the structure and function of primary cilia in liver transplant recipients that develop biliary complications (n = 7) in comparison with recipients without biliary complications (n = 12). To study the biological effects of the primary cilia during transplantation, we generated murine models that recapitulate liver procurement and cold storage, and assessed the elimination of the primary cilia in biliary epithelial cells in the K19CreER T Kif3a fl /fl mouse model. To explore the molecular mechanisms responsible for the observed phenotypes we used in vitro models of ischemia, cellular senescence and primary cilia ablation. Finally, we used pharmacological and genetic approaches to target cellular senescence and the primary cilia, both in mouse models and discarded human donor livers., Results: Prolonged ischemic periods before transplantation result in ciliary shortening and cellular senescence, an irreversible cell cycle arrest that blocks regeneration. Our results indicate that primary cilia damage results in biliary injury and a loss of regenerative potential. Senescence negatively impacts primary cilia structure and triggers a negative feedback loop that further impairs regeneration. Finally, we explore how targeted interventions for cellular senescence and/or the stabilisation of the primary cilia improve biliary regeneration following ischemic injury., Conclusions: Primary cilia play an essential role in biliary regeneration and we demonstrate that senolytics and cilia-stabilising treatments provide a potential therapeutic opportunity to reduce the rate of biliary complications and improve clinical outcomes in liver transplantation., Impact and Implications: Up to 25% of liver transplants result in biliary complications, leading to additional surgery, retransplants, or death. We found that the incidence of biliary complications is increased by damage to the primary cilium, an antenna that protrudes from the cell and is key to regeneration. Here, we show that treatments that preserve the primary cilia during the transplant process provide a potential solution to reduce the rates of biliary complications., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background & Aims: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFβ signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct., Methods: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning., Results: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation., Conclusions: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture., Impact and Implications: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed., (Copyright © 2024. Published by Elsevier B.V.)

Objective: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance., Design: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data., Results: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours., Conclusions: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings., Competing Interests: Competing interests: JBA is a member of the scientific advisory board at SEALD, Norway and reports scientific consultancies for QED Therapeutics and Flagship Pioneering. JBA has received research funding from Incyte. CB received honoraria as speaker (Astrazeneca, Incyte) and consultant (Incyte, Servier, Boehringer Ingelheim, Astrazeneca), received research funds (Avacta, Medannex, Servier) and her spouse is an employee of Astrazeneca. JML is receiving research support from Eisai, Bayer HealthCare Pharmaceuticals, Ipsen, and consulting fees from Eisai, Merck, Bristol-Myers Squibb, Eli Lilly, Roche, Genentech, Ipsen, Glycotest, AstraZeneca, Bayer HealthCare Pharmaceuticals, Omega Therapeutics, Mina Alpha, Boston Scientific, Exelixis, Bluejay and Captor Therapeutics. RM has received consulting and lecture fees from Servier and Roche and travel and education funding from MSD, Eli Lilly, Bayer, Roche, Astrazeneca. AL reports receiving consulting fees from Intercept Pharma, Alfa Sigma, Takeda, and Albireo Pharma, and speakers’ fees from Gilead, Abbvie, MSD, Intercept Pharma, AlfaSigma, GSK and Incyte., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)

Background: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities., Methods: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay., Findings: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness., Interpretation: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC., Funding: Foundation for Liver Research, London., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35 ) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-β (TGFβ) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFβ signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

The Notch signalling pathway is an evolutionarily conserved mechanism of cell-cell communication that mediates cellular proliferation, fate determination and maintenance of stem/progenitor cell populations across tissues. Although it was originally identified as a critical regulator of embryonic liver development, NOTCH signalling activation has been associated with the pathogenesis of a number of paediatric and adult liver diseases. It remains unclear, however, what role NOTCH actually plays in these pathophysiological processes and whether NOTCH activity represents the reactivation of a conserved developmental programme that is essential for adult tissue repair. In this Review, we explore the concepts that NOTCH signalling reactivation in the biliary epithelium is a reiterative and essential response to bile duct damage and that, in disease contexts in which biliary epithelial cells need to be regenerated, NOTCH signalling supports ductular regrowth. Furthermore, we evaluate the recent literature on NOTCH signalling as a critical factor in progenitor-mediated hepatocyte regeneration, which indicates that the mitogenic role for NOTCH signalling in biliary epithelial cell proliferation has also been co-opted to support other forms of epithelial regeneration in the adult liver., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Farley, Chengrui, Palmer, Liu, Kousa, Rouse, Major, Sweetman, Morys, Corsinotti, Nichols, Ure, McLay, Boulter, Chapman, Tomlinson and Blackburn.)

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA., (© 2023. Springer Nature Limited.)

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1 + ) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1 + cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1 + cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D ( Ubd ) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1 + cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8 -expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1 + cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Risk-stratified follow-up for endometrial cancer (EC) is being introduced in many cancer centres; however, there appears to be diversity in the structure and availability of schemes across the UK. This study aimed to investigate clinicians' and clinical specialist nurses' (CNS) experiences of follow-up schemes for EC, including patient-initiated follow-up (PIFU), telephone follow-up (TFU) and clinician-led hospital follow-up (HFU). A mixed-methods study was conducted, consisting of an online questionnaire to CNSs, an audience survey of participants attending a national "Personalising Endometrial Cancer Follow-up" educational meeting, and qualitative semi-structured telephone interviews with clinicians involved in the follow-up of EC. Thematic analysis identified three main themes to describe clinicians' views: appropriate patient selection; changing from HFU to PIFU schemes; and the future of EC follow-up schemes. Many participants reported that the COVID-19 pandemic impacted EC follow-up by accelerating the transition to PIFU/TFU. Overall, there was increasing support for non-HFU schemes for patients who have completed primary treatment of EC; however, barriers were identified for non-English-speaking patients and those who had communication challenges. Given the good long-term outcome associated with EC, greater focus is needed to develop resources to support patients post-treatment and individualise follow-up according to patients' personal needs and preferences.