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Intrahepatic cholangiocarcinoma is the second most frequent primary liver cancer after hepatocellular carcinoma. According to International Classification of Diseases-11 (ICD-11), intrahepatic cholangiocarcinoma is identified by a specific diagnostic code, different with respect to perihilar-CCA or distal-CCA. Intrahepatic cholangiocarcinoma originates from intrahepatic small or large bile ducts including the second-order bile ducts and has a silent presentation that combined with the highly aggressive nature and refractoriness to chemotherapy contributes to the alarming increasing incidence and mortality. Indeed, at the moment of the diagnosis, less than 40% of intrahepatic cholangiocarcinoma are suitable of curative surgical therapy, that is so far the only effective treatment. The main goals of clinicians and researchers are to make an early diagnosis, and to carry out molecular characterization to provide the patient with personalized treatment. Unfortunately, these goals are not easily achievable because of the heterogeneity of this tumor from anatomical, molecular, biological, and clinical perspectives. However, recent progress has been made in molecular characterization, surgical treatment, and management of intrahepatic cholangiocarcinoma and, this article deals with these advances., Competing Interests: Conflict of interest I declare that neither I nor the co-authors, Rosanna Venere, Emanuela Ribichini, Francesco Covotta, Vincenz Cardinale and Domenico Alvaro have a conflict of interest regarding the review entitled “Intrahepatic cholangiocarcinoma: evolving strategies in management and treatment”, (Copyright © 2023 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Patients with chronic gastrointestinal disease such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), celiac disease, gastroesophageal reflux disease (GERD), pancreatitis, and chronic liver disease (CLD) often suffer from obesity because of coincidence (IBD, IBS, celiac disease) or related pathophysiology (GERD, pancreatitis and CLD). It is unclear if such patients need a particular diagnostic and treatment that differs from the needs of lean gastrointestinal patients. The present guideline addresses this question according to current knowledge and evidence., Objective: The present practical guideline is intended for clinicians and practitioners in general medicine, gastroenterology, surgery and other obesity management, including dietitians and focuses on obesity care in patients with chronic gastrointestinal diseases., Methods: The present practical guideline is the shortened version of a previously published scientific guideline developed according to the standard operating procedure for ESPEN guidelines. The content has been re-structured and transformed into flow-charts that allow a quick navigation through the text., Results: In 100 recommendations (3× A, 33× B, 24 × 0, 40× GPP, all with a consensus grade of 90% or more) care of gastrointestinal patients with obesity - including sarcopenic obesity - is addressed in a multidisciplinary way. A particular emphasis is on CLD, especially metabolic associated liver disease, since such diseases are closely related to obesity, whereas liver cirrhosis is rather associated with sarcopenic obesity. A special chapter is dedicated to obesity care in patients undergoing bariatric surgery. The guideline focuses on adults, not on children, for whom data are scarce. Whether some of the recommendations apply to children must be left to the judgment of the experienced pediatrician., Conclusion: The present practical guideline offers in a condensed way evidence-based advice how to care for patients with chronic gastrointestinal diseases and concomitant obesity, an increasingly frequent constellation in clinical practice., (Copyright © 2023 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: Common precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum., Methods: Human duodena were obtained from organ donors, and duodenal submucosal gland cells were isolated after removal of the mucosa layer. Cells were cultured on plastic or as organoids and were transplanted into severe combined immunodeficient (SCID) mouse livers., Results: In situ studies of submucosal glands in the human duodenum revealed cells expressing stem/progenitor cell markers that had unique phenotypic traits distinguishable from intestinal crypt cells. Genetic signature studies indicated that the cells are closer to biliary tree stem cells and to definitive endodermal cells than to adult hepatocytes, supporting the interpretation that they are endodermal stem/progenitor cells. In vitro, human duodenal submucosal gland cells demonstrated clonal growth, capability to form organoids, and ability to acquire functional hepatocyte traits. In vivo, transplanted cells engrafted into the livers of immunocompromised mice and differentiated to mature liver cells. In an experimental model of fatty liver, human duodenal submucosal gland cells were able to rescue hosts from liver damage by supporting repopulation and regeneration of the liver., Conclusions: A cell population with clonal growth and organoid formation capability, which has liver differentiation potency in vitro and in vivo in murine experimental models, is present within adult duodenal submucosal glands. These cells can be isolated, do not require reprogramming, and thus could potentially represent a novel cell source for regenerative medicine of the liver., Impact and Implications: Cell therapies for liver disease could represent an option to support liver function, but the identification of sustainable and viable cell sources is critical. Here, we describe a cell population with organoid formation capability and liver-specific regenerative potential in submucosal glands of the human duodenum. Duodenal submucosal gland cells are isolated from adult organs, do not require reprogramming, and could rescue hepatocellular damage in preclinical models of chronic, but not acute, liver injury. Duodenal submucosal gland cells could represent a potential candidate cell source for regenerative medicine of the liver, but the determination of cell dose and toxicity is needed before clinical testing in humans., Competing Interests: Conflicts of interest The authors declare no competing interests, but with the following exceptions. GC, EG, DA, VC, and LMR are inventors on patents on the following: isolation methods and ex vivo maintenance of biliary tree stem cells (US Patent No. US20110135610A1); cryopreservation methods for biliary tree stem cells (US Patent No. US20180295834A1); grafting methods for stem cells (US Patent Nos. 9533013 and 9750770); isolation methods for duodenal submucosal gland cells (US Patent Application 20190300849A1). WZ and LMR are inventors on a patent for patch grafting methods for organoids of stem/progenitors into solid organs (US Patent No. US20180361027A1). The IP is owned by Sapienza University of Rome and/or by the University of North Carolina. The license for the clinical uses in humans is owned by Vesta Therapeutics, and that for the veterinary uses and the non-clinical, commercial uses by PhoenixSongs Biologicals (Branford, CT, USA). None of the authors are employees, receive compensation, or have equity positions in Vesta Therapeutics or PhoenixSongs Biologicals. The exception is LMR, who has a founder’s equity position in PhoenixSongs Biologicals but has received no pay, royalties, or other form of compensation from the company. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2022 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Patients with chronic gastrointestinal (GI) disease such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), celiac disease, gastroesophageal reflux disease (GERD), pancreatitis, and chronic liver disease (CLD) often suffer from obesity because of coincidence (IBD, IBS, celiac disease) or related pathophysiology (GERD, pancreatitis and CLD). It is unclear if such patients need a particular diagnostic and treatment that differs from the needs of lean GI patients. The present guideline addresses this question according to current knowledge and evidence., Objective: The objective of the guideline is to give advice to all professionals working in the field of gastroenterology care including physicians, surgeons, dietitians and others how to handle patients with GI disease and obesity., Methods: The present guideline was developed according to the standard operating procedure for ESPEN guidelines, following the Scottish Intercollegiate Guidelines Network (SIGN) grading system (A, B, 0, and good practice point (GPP)). The procedure included an online voting (Delphi) and a final consensus conference., Results: In 100 recommendations (3x A, 33x B, 24x 0, 40x GPP, all with a consensus grade of 90% or more) care of GI patients with obesity - including sarcopenic obesity - is addressed in a multidisciplinary way. A particular emphasis is on CLD, especially fatty liver disease, since such diseases are closely related to obesity, whereas liver cirrhosis is rather associated with sarcopenic obesity. A special chapter is dedicated to obesity care in patients undergoing bariatric surgery. The guideline focuses on adults, not on children, for whom data are scarce. Whether some of the recommendations apply to children must be left to the judgment of the experienced pediatrician., Conclusion: The present guideline offers for the first time evidence-based advice how to care for patients with chronic GI diseases and concomitant obesity, an increasingly frequent constellation in clinical practice., Competing Interests: Conflict of interest The expert members of the working group were accredited by the ESPEN Guidelines Group, the ESPEN Education and Clinical Practice Committee, the ESPEN executive, and the UEG Quality of Care Task Force. All expert members have declared their individual conflicts of interest according to the rules of the International Committee of Medical Journal Editors (ICMJE). If potential conflicts were indicated, they were reviewed by the ESPEN guideline officers and, in cases of doubts, by the ESPEN executive. None of the expert panel had to be excluded from the working group or from co-authorship because of serious conflicts. The conflict-of-interest forms are stored at the ESPEN guideline office and can be reviewed with legitimate interest upon request to the ESPEN executive., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background & Aims: Cholangiocarcinoma (CCA) is a rare and heterogeneous biliary cancer, whose incidence and related mortality is increasing. This study investigates the clinical course of CCA and subtypes (intrahepatic [iCCA], perihilar [pCCA], and distal [dCCA]) in a pan-European cohort., Methods: The ENSCCA Registry is a multicenter observational study. Patients were included if they had a histologically proven diagnosis of CCA between 2010-2019. Demographic, histomorphological, biochemical, and clinical studies were performed., Results: Overall, 2,234 patients were enrolled (male/female=1.29). iCCA (n = 1,243) was associated with overweight/obesity and chronic liver diseases involving cirrhosis and/or viral hepatitis; pCCA (n = 592) with primary sclerosing cholangitis; and dCCA (n = 399) with choledocholithiasis. At diagnosis, 42.2% of patients had local disease, 29.4% locally advanced disease (LAD), and 28.4% metastatic disease (MD). Serum CEA and CA19-9 showed low diagnostic sensitivity, but their concomitant elevation was associated with increased risk of presenting with LAD (odds ratio 2.16; 95% CI 1.43-3.27) or MD (odds ratio 5.88; 95% CI 3.69-9.25). Patients undergoing resection (50.3%) had the best outcomes, particularly with negative-resection margin (R0) (median overall survival [mOS] = 45.1 months); however, margin involvement (R1) (hazard ratio 1.92; 95% CI 1.53-2.41; mOS = 24.7 months) and lymph node invasion (hazard ratio 2.13; 95% CI 1.55-2.94; mOS = 23.3 months) compromised prognosis. Among patients with unresectable disease (49.6%), the mOS was 10.6 months for those receiving active palliative therapies, mostly chemotherapy (26.2%), and 4.0 months for those receiving best supportive care (20.6%). iCCAs were associated with worse outcomes than p/dCCAs. ECOG performance status, MD and CA19-9 were independent prognostic factors., Conclusion: CCA is frequently diagnosed at an advanced stage, a proportion of patients fail to receive cancer-specific therapies, and prognosis remains dismal. Identification of preventable risk factors and implementation of surveillance in high-risk populations are required to decrease cancer-related mortality., Lay Summary: This is, to date, the largest international (pan-European: 26 hospitals and 11 countries) observational study, in which the course of cholangiocarcinoma has been investigated, comparing the 3 subtypes based on the latest International Classification of Diseases 11 th Edition (ICD-11) (i.e., intrahepatic [2C12], perihilar [2C18], or distal [2C15] affected bile ducts), which come into effect in 2022. General and tumor-type specific features at diagnosis, risk factors, biomarker accuracy, as well as patient management and outcomes, are presented and compared, outlining the current clinical state of cholangiocarcinoma in Europe., Competing Interests: Conflict of interest AF declares lecture fees (from Bayer HealthCare, Gilead, Roche, MSD and Boston Scientific), and consultancy fees (from Bayer HealthCare, AstraZeneca, Roche, Guerbert, SIRTEX and Exact Science). AL reported travel and educational support (from Ipsen, Pfizer, Bayer, Advanced Acceletaro Applications, SirtEx, Novartis, Mylan and Delcath), speaker honoraria (from Merck, Pfizer, Ipsen, Incyte and AAA), advisory honoraria (from EISAI, Nutricia Ipsen, QED and Roche), and she is Member of the Knowledge Network and NETConnect Initiatives funded by Ipsen. ALl declares lecture fees (from Intercept Pharma, Abbvie, Gilead, AlfaSigma, and MSD) and consultancy fees (from Intercept and Alfa Sigma). AL-M declares travel and educational support (from Pfizer, Roche, Sanofi, Rovi, Pharma Mar and Merck). CB declares consultancy honoraria (from Incyte). GV declares consultancy fee (from Bayer HealthCare). JBA declares consulting role (for QED Therapeutics and SEALD). JMB declares research grants (from Incyte), personal fees for lecturer (from Bayer and Intercept), and consulting role (for QED Therapeutics, Albireo Pharma and OWL Metabolomics). JWV reports personal fees (from Agios, AstraZeneca, Baxter, Genoscience Pharma, Hutchison Medipharma, Imaging Equipment Ltd (AAA), Incyte, Ipsen, Mundipharma EDO, Mylan, QED, Servier, Sirtex, Zymeworks), and grants, personal fees and non-financial support (from NuCana). LF declares research grants (from Progetto di Ricerca Dipartimentale), honoraria (from Obesity 4C Complex Clinical Cases Contest), leadership role (for HCERES, NCN), and travel expenses (from AASLD, AISF and EASL). MP-S declares honoraria (from Roche and Servier), consulting or advisory role (for AstraZeneca) and travel expenses (from Roche, BMS and Incyte). KU declares fees for advisory role (BMS). ALC, AS, ASc, AS-L, AV, BGK, DA, FL-L, GC, GLG, HJK, JA, JI, JIE, JJGM, JK, JPJ, KE, LB, LI-S, LK, MK, MM, NJ, RB, RIRM, SB, TF, VC, VS, WH, and ZS declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The fetal liver is unique because of the coexistence of cells with endodermal and mesenchymal origins, making it a potential source of hepatic and pancreatic regenerative medicine. The liver appears at about the third week of gestation, growing rapidly from the fifth to the 10th week. We define fetal liver from 10 weeks of gestation, when hematopoietic progenitor cells gradually migrate from the aorta-mesonephros-gonad region to colonize the liver. Indeed, the fetal liver may be the most available source of cell therapy for liver disease. We conducted a review of the literature using Medline and EMBASE (up to May 2021) to identify clinical studies in which patients with liver disease had been given fetal liver cell therapy. This literature review highlighted the heterogeneity of cell isolation and selection protocols, which hinders the ability to pool data and perform a meta-analysis. A limitation of the studies analyzed was the scarcity of reports (n = 8) and the extremely small sample sizes (median sample size of treated patients was two), although there was a fairly long follow-up (median 12 months). The weeks after conception ranged from 16 to 34. There were no randomized controlled trials, and therefore no study was stratified as being of good methodological quality. Cryopreservation may help to circumvent the critical logistic issues that hamper the use of fetal liver cell therapy in clinical practice. To help consolidate the role of the fetal liver in regenerative medicine, good preclinical translational studies are necessary, whereas tracing strategies and biopsy-based endpoints are crucial in the clinic, along with well-designed, large, multicenter, randomized controlled trials using clinically applicable primary outcomes and refined imaging assessment., Competing Interests: Declarations of Competing Interest The authors report no conflicts of interest., (Copyright © 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The microenvironment of intrahepatic cholangiocarcinoma (iCCA) is hypovascularized, with an extensive lymphatic network. This leads to rapid cancer spread into regional lymph nodes and the liver parenchyma, precluding curative treatments. Herein, we investigated which factors released in the iCCA stroma drive the inhibition of angiogenesis and promote lymphangiogenesis., Methods: Quantitative proteomics was performed on extracellular fluid (ECF) proteins extracted both from cancerous and non-cancerous tissues (NCT) of patients with iCCA. Computational biology was applied on a proteomic dataset to identify proteins involved in the regulation of vessel formation. Endothelial cells incubated with ECF from either iCCA or NCT specimens were used to assess the role of candidate proteins in 3D vascular assembly, cell migration, proliferation and viability. Angiogenesis and lymphangiogenesis were further investigated in vivo by a heterotopic transplantation of bone marrow stromal cells, along with endothelial cells in SCID/beige mice., Results: Functional analysis of upregulated proteins in iCCA unveils a soluble angio-inhibitory milieu made up of thrombospondin (THBS)1, THBS2 and pigment epithelium-derived factor (PEDF). iCCA ECF was able to inhibit in vitro vessel morphogenesis and viability. Antibodies blocking THBS1, THBS2 and PEDF restored tube formation and endothelial cell viability to levels observed in NCT ECF. Moreover, in transplanted mice, the inhibition of blood vessel formation, the de novo generation of the lymphatic network and the dissemination of iCCA cells in lymph nodes were shown to depend on THBS1, THBS2 and PEDF expression., Conclusions: THBS1, THBS2 and PEDF reduce blood vessel formation and promote tumor-associated lymphangiogenesis in iCCA. Our results identify new potential targets for interventions to counteract the dissemination process in iCCA., Lay Summary: Intrahepatic cholangiocarcinoma is a highly aggressive cancer arising from epithelial cells lining the biliary tree, characterized by dissemination into the liver parenchyma via lymphatic vessels. Herein, we show that the proteins THBS1, THBS2 and PEDF, once released in the tumor microenvironment, inhibit vascular growth, while promoting cancer-associated lymphangiogenesis. Therefore, targeting THBS1, THBS2 and PEDF may be a promising strategy to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of intrahepatic cholangiocarcinoma., Competing Interests: Conflict of interest The authors of this study have no conflict of interest to declare. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The microbiota-gut-liver-lung axis plays a bidirectional role in the pathophysiology of a number of infectious diseases. During the course of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and 2 (SARS-CoV-2) infection, this pathway is unbalanced due to intestinal involvement and systemic inflammatory response. Moreover, there is convincing preliminary evidence linking microbiota-gut-liver axis perturbations, proinflammatory status, and endothelial damage in noncommunicable preventable diseases with coronavirus disease 2019 (Covid-19) severity. Intestinal damage due to SARS-CoV-2 infection, systemic inflammation-induced dysfunction, and IL-6-mediated diffuse vascular damage may increase intestinal permeability and precipitate bacterial translocation. The systemic release of damage- and pathogen-associated molecular patterns (e.g. lipopolysaccharides) and consequent immune-activation may in turn auto-fuel vicious cycles of systemic inflammation and tissue damage. Thus, intestinal bacterial translocation may play an additive/synergistic role in the cytokine release syndrome in Covid-19. This review provides evidence on gut-liver axis involvement in Covid-19 as well as insights into the hypothesis that intestinal endotheliitis and permeability changes with bacterial translocation are key pathophysiologic events modulating systemic inflammatory response. Moreover, it presents an overview of readily applicable measures for the modulation of the gut-liver axis and microbiota in clinical practice., Competing Interests: Declaration of Competing Interest None, (Copyright © 2020 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background: Due to significant limitations to the access to orthotropic liver transplantation, cell therapies for liver diseases have gained large interest worldwide., Scope of Review: To revise current literature dealing with cell therapy for liver diseases. We discussed the advantages and pitfalls of the different cell sources tested so far in clinical trials and the rationale underlying the potential benefits of transplantation of human biliary tree stem cells (hBTSCs)., Major Conclusions: Transplantation of adult hepatocytes showed transient benefits but requires immune-suppression that is a major pitfall in patients with advanced liver diseases. Mesenchymal stem cells and hematopoietic stem cells transplanted into patients with liver diseases are not able to replace resident hepatocytes but rather they target autoimmune or inflammatory processes into the liver. Stem cells isolated from fetal or adult liver have been recently proposed as alternative cell sources for advanced liver cirrhosis and metabolic liver disease. We demonstrated the presence of multipotent cells expressing a variety of endodermal stem cell markers in (peri)-biliary glands of bile ducts in fetal or adult human tissues, and in crypts of gallbladder epithelium. In the first cirrhotic patients treated in our center with biliary tree stem cell therapy, we registered no adverse event but significant benefits., General Significance: The biliary tree stem cell could represent the ideal cell source for the cell therapy of liver diseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are human primary cholangiopathies characterized by the damage of mature cholangiocytes and by the appearance of ductular reaction (DR) as the results of hepatic progenitor cell activation. This study evaluated the differences in progenitor cell niche activation between these two cholangiopathies. Liver tissue was obtained from healthy liver donors (n = 5) and from patients with PSC (n = 20) or PBC (n = 20). DR, progenitor cell phenotype, and signaling pathways were investigated by IHC analysis and immunofluorescence. Our results indicated that DR was more extended, appeared earlier, and had a higher proliferation index in PBC compared with PSC. In PBC, DR was strongly correlated with clinical prognostic scores. A higher percentage of sex determining region Y-box (SOX)9 + and cytokeratin 19 + cells but fewer features of hepatocyte fate characterized progenitor cell activation in PBC versus PSC. Lower levels of laminin and neurogenic locus notch homolog protein 1 but higher expression of wingless-related integration site (WNT) family pathway components characterize progenitor cell niche in PSC compared with PBC. In conclusion, progenitor cell activation differs between PSC and PBC and is characterized by a divergent fate commitment and different signaling pathway predominance. In PBC, DR represents a relevant histologic prognostic marker., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche., Methods: CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14 + with CCA-sphere conditioned medium., Results: CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p=0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14 + macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163 + set was found in the tumor front of human CCA specimens (n=23) and correlated with a high level of serum cancer antigen 19.9 (n=17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n=12). Surprisingly, a significant association of IL13, IL34 and osteoactivin with sphere stem-like genes was provided by a CCA database (n=104). In vitro combination of IL13, IL34, osteoactivin was responsible for macrophage-differentiation and invasion, as well as for in vivo tumor-promoting effect., Conclusion: CCA-CSCs molded a specific subset of stem-like associated macrophages thus providing a rationale for a synergistic therapeutic strategy for CCA-disease., Lay Summary: Immune plasticity represents an important hallmark of tumor outcome. Since cancer stem cells are able to manipulate stromal cells to their needs, a better definition of the key dysregulated immune subtypes responsible for cooperating in supporting tumor initiation may facilitate the development of new therapeutic approaches. Considering that human cholangiocarcinoma represents a clinical emergency, it is essential to move to predictive models in order to understand the adaptive process of macrophage component (imprinting, polarization and maintenance) engaged by tumor stem-like compartment., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Primary sclerosing cholangitis (PSC) is characterised by fibro-stenosing strictures involving extrahepatic and/or large intrahepatic bile ducts. Mechanisms leading to bile duct injury are poorly understood. We aimed to study the biliary tree stem cell compartment located in peribiliary glands of extrahepatic and large intrahepatic bile ducts and its role in the pathogenesis of biliary fibrosis in PSC., Methods: Specimens containing extrahepatic or large intrahepatic bile ducts were obtained from normal liver (n=6), liver explants from patients with PSC (n=11), and primary biliary cirrhosis (n=6). Specimens were processed for histology, immunohistochemistry and immunofluorescence., Results: In PSC samples, progressive hyperplasia and mucinous metaplasia of peribiliary glands were observed in large ducts with fibrosis, but not in inflamed ducts without fibrosis. Peribiliary gland hyperplasia was associated with progressive biliary fibrosis and the occurrence of dysplastic lesions. Hyperplasia of peribiliary glands was determined by the expansion of biliary tree stem cells, which sprouted towards the surface epithelium. In PSC, peribiliary glands and myofibroblasts displayed enhanced expression of Hedgehog pathway components. Peribiliary glands in ducts with onion skin-like fibrosis expressed epithelial-to-mesenchymal transition traits associated with components of Hedgehog pathway, markers of senescence and autophagy., Conclusions: The biliary tree stem cell compartment is activated in PSC, its activation contributes to biliary fibrosis, and is sustained by the Hedgehog pathway. Our findings suggest a key role for peribiliary glands in the progression of bile duct lesions in PSC and could explain the associated high risk of cholangiocarcinoma., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Cholangiocarcinomas (CCAs) comprise a mucin-secreting form, intrahepatic or perihilar, and a mixed form located peripherally. We characterized cancer stem cells (CSCs) in CCA subtypes and evaluated their cancerogenic potential. CSC markers were investigated in 25 human CCAs in primary cultures and established cell lines. Tumorigenic potential was evaluated in vitro or in xenografted mice after s.c. or intrahepatic injection in normal and cirrhotic (carbon tetrachloride-induced) mice. CSCs comprised more than 30% of the tumor mass. Although the CSC profile was similar between mucin-intrahepatic and mucin-perihilar subtypes, CD13(+) CSCs characterized mixed-intrahepatic, whereas LGR5(+) characterized mucin-CCA subtypes. Many neoplastic cells expressed epithelial-mesenchymal transition markers and coexpressed mesenchymal and epithelial markers. In primary cultures, epithelial-mesenchymal transition markers, mesenchymal markers (vimentin, CD90), and CD13 largely predominated over epithelial markers (CD133, EpCAM, and LGR5). In vitro, CSCs expressing epithelial markers formed a higher number of spheroids than CD13(+) or CD90(+) CSCs. In s.c. tumor xenografts, tumors dominated by stromal markers were formed primarily by CD90(+) and CD13(+) cells. By contrast, in intrahepatic xenografts in cirrhotic livers, tumors were dominated by epithelial traits reproducing the original human CCAs. In conclusion, CSCs were rich in human CCAs, implicating CCAs as stem cell-based diseases. CSC subpopulations generate different types of cancers depending on the microenvironment. Remarkably, CSCs reproduce the original human CCAs when injected into cirrhotic livers., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Human biliary tree stem/progenitor cells (hBTSCs) are multipotent epithelial stem cells, easily obtained from the biliary tree, with the potential for regenerative medicine in liver, biliary tree, and pancreas diseases. Recent reports indicate that human mesenchymal stem cells are able to modulate the T cell immune response. However, no information exists on the capabilities of hBTSCs to control the allogeneic response. The aims of this study were to evaluate FasL expression in hBTSCs, to study the in vitro interaction between hBTSCs and human lymphocytes, and the role of Fas/FasL modulation in inducing T cell apoptosis in hBTSCs/T cell co-cultures., Methods: Fas and FasL expression were evaluated in situ and in vitro by immunofluorescence and western blotting. Co-cultures of hBTSCs with human leukocytes were used to analyze the influence of hBTSCs on lymphocytes activation and apoptosis., Results: hBTSCs expressed HLA antigens and FasL in situ and in vitro. Western blot data demonstrated that hBTSCs constitutively expressed high levels of FasL that increased after co-culture with T cells. Confocal microscopy demonstrated that FasL expression was restricted to EpCAM(+)/LGR5(+) cells. FACS analysis of T cells co-cultured with hBTSCs indicated that hBTSCs were able to induce apoptosis in activated CD4(+) and CD8(+) T cell populations. Moreover, the Fas receptor appears to be more expressed in T cells co-cultured with hBTSCs than in resting T cells., Conclusions: Our data suggest that hBTSCs could modulate the T cell response through the production of FasL, which influences the lymphocyte Fas/FasL pathway by inducing "premature" apoptosis in CD4(+) and CD8(+) T cells., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Multipotent stem/progenitor cells are found in peribiliary glands throughout human biliary trees and are able to generate mature cells of hepato-biliary and pancreatic endocrine lineages. The presence of endodermal stem/progenitors in human gallbladder was explored., Methods: Gallbladders were obtained from organ donors and laparoscopic surgery for symptomatic cholelithiasis. Tissues or isolated cells were characterized by immunohistochemistry and flow cytometry. EpCAM+ (Epithelial Cell Adhesion Molecule) cells were immunoselected by magnetic microbeads, plated onto plastic in self-replication conditions and subsequently transferred to distinct serum-free, hormonally defined media tailored for differentiation to specific adult fates. In vivo studies were conducted in an experimental model of liver cirrhosis., Results: The gallbladder does not have peribiliary glands, but it has stem/progenitors organized instead in mucosal crypts. Most of these can be isolated by immune-selection for EpCAM. Approximately 10% of EpCAM+ cells in situ and of immunoselected EpCAM+ cells co-expressed multiple pluripotency genes and various stem cell markers; other EpCAM+ cells qualified as progenitors. Single EpCAM+ cells demonstrated clonogenic expansion ex vivo with maintenance of stemness in self-replication conditions. Freshly isolated or cultured EpCAM+ cells could be differentiated to multiple, distinct adult fates: cords of albumin-secreting hepatocytes, branching ducts of secretin receptor+ cholangiocytes, or glucose-responsive, insulin/glucagon-secreting neoislets. EpCAM+ cells transplanted in vivo in immune-compromised hosts gave rise to human albumin-producing hepatocytes and to human Cytokeratin7+ cholangiocytes occurring in higher numbers when transplanted in cirrhotic mice., Conclusions: Human gallbladders contain easily isolatable cells with phenotypic and biological properties of multipotent, endodermal stem cells., (Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Biliary tree, liver, and pancreas share a common embryological origin. We previously demonstrated the presence of stem/progenitor cells of endodermal origin in the adult human extrahepatic biliary tree. This study evaluated the human foetal biliary trees as sources of stem/progenitor cells of multiple endodermal-derived mature fates., Methods: Human foetal intrahepatic and extrahepatic biliary tree tissues and isolated cells were tested for cytoplasmic and surface markers of stem cells and committed progenitors, as well as endodermal transcription factors requisite for a liver versus pancreatic fate. In vitro and in vivo experiments were conducted to evaluate the potential mature fates of differentiation., Results: Foetal biliary tree cells proliferated clonogenically for more than 1 month on plastic in a serum-free Kubota medium. After culture expansion, cells exhibited multipotency and could be restricted to certain lineages under defined microenvironments, including hepatocytes, cholangiocytes, and pancreatic islet cells. Transplantation of foetal biliary tree cells into the livers of immunodeficient mice resulted in effective engraftment and differentiation into mature hepatocytes and cholangiocytes., Conclusions: Foetal biliary trees contain multipotent stem/progenitor cells comparable with those in adults. These cells can be easily expanded and induced in vitro to differentiate into liver and pancreatic mature fates, and engrafted and differentiated into mature cells when transplanted in vivo. These findings further characterise the development of these stem/progenitor cell populations from foetuses to adults, which are thought to contribute to liver and pancreas organogenesis throughout life., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Polycystin-1 and -2 (PC-1 and PC-2) are critical components of primary cilia, which act as mechanosensors and drive cell response to injury. PC-1 activation involves the cleavage/processing of PC-1 cytoplasmic tail, driven by regulated intramembrane proteolysis or ubiquitine/proteasome, translocation in the nucleus and activation of transcription factors. Mutations of PC-1 or PC-2 occur in polycystic liver where cholangiocyte proliferation is enhanced., Aim: We evaluated the involvement of PC-1 and PC-2 in modulating cholangiocyte proliferation., Methods: We investigated rat cholangiocytes induced to proliferate by 17beta-oestradiol. Proliferation was evaluated by PCNA immunoblotting or [(3)H]-thymidine incorporation into DNA. PC-1 silencing was performed by siRNA, while inhibition of regulated intramembrane proteolysis or proteasome by gamma-secretase inhibitor, leupeptin or MG115., Results: Cholangiocyte proliferation was associated with decreased PC-1 and PC-2 expression, which was inversely correlated with enhanced PCNA. The selective silencing of PC-1 induced activation of cholangiocyte proliferation in association with decreased PC-1 expression. Two different regulated intramembrane proteolysis inhibitors, gamma-secretase-inhibitor and leupeptin, and the proteasome inhibitor, MG115, abolished the 17beta-oestradiol proliferative effect., Conclusions: PC-1 and PC-2 play a major role as modulators of cholangiocyte proliferation suggesting that primary cilia may act as sensors of cell injury driving, when activated, a proliferative cholangiocyte response to trigger the reparative processes., (Copyright 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [(3)H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17beta-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17beta-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism.