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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.)

Objectives: Early diagnosis of biliary atresia is essential to improve long-term outcomes. Newborn screening with an infant stool color card allows early recognition of biliary atresia patients. Our aim was to develop and validate a mobile phone application (PopòApp) able to identify acholic stools., Methods: An intuitive app was developed for iOS and Android smartphones. A learning machine process was used to generate an algorithm for stools color recognition based on the seven colors of the infant stool color card, which were considered as the gold standard. Consecutive images of stools were taken by the PopòApp, directly into the diapers of children aged ≤6 months. The PopòApp classified the photographs as "normal", "acholic" or "uncertain". To validate the PopòApp, four doctors independently classified all images, and only those for which all doctors agreed were included. The sensitivity, specificity, positive/negative predictive values, and accuracy of the PopòApp were evaluated., Results: Of 165 images collected, 160 were included in the study. All acholic stools were recognized by the PopòApp. The PopòApp sensitivity was 100% (95% CI:93.9%-100%) with no false negatives, regardless of the brand of phone. The specificity was 99.0% (95% CI:94.6%-99.9%). The accurancy of the PopòApp was 99.4% (95% CI:96.6%-99.9%), with a positive predictive value of 98.4% (95% CI:89.8%-99.8%)., Conclusion: The current study proved, in a large cohort, that the PopòApp is an accurate and easy tool for recognition of acholic stools. The mobile App may represent an effective strategy for the early referral of children with acholic stools, and potentially could improve the outcomes of biliary atresia.

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA)., Approach and Results: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine-rich repeat-containing G protein-coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real-time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real-time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme-linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCA LGR5+ and in iCCA CD133+ cells compared with unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in a dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls., Conclusions: DCLK1 characterizes a specific CSC subpopulation of iCCA CD133+ and pCCA LGR5+ , and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis., (© 2020 by the American Association for the Study of Liver Diseases.)

In normal human livers, EpCAM pos cells are mostly restricted in two distinct niches, which are (i) the bile ductules and (ii) the mucous glands present inside the wall of large intrahepatic bile ducts (the so-called peribiliary glands). These EpCAM pos cell niches have been proven to harbor stem/progenitor cells with great importance in liver and biliary tree regeneration and in the pathophysiology of human diseases. The EpCAM pos progenitor cells within bile ductules are engaged in driving regenerative processes in chronic diseases affecting hepatocytes or interlobular bile ducts. The EpCAM pos population within peribiliary glands is activated when regenerative needs are finalized to repair large intra- or extra-hepatic bile ducts affected by chronic pathologies, including primary sclerosing cholangitis and ischemia-induced cholangiopathies after orthotopic liver transplantation. Finally, the presence of distinct EpCAM pos cell populations may explain the histological and molecular heterogeneity characterizing cholangiocarcinoma, based on the concept of multiple candidate cells of origin. This review aimed to describe the precise anatomical distribution of EpCAM pos populations within the liver and the biliary tree and to discuss their contribution in the pathophysiology of human liver diseases, as well as their potential role in regenerative medicine of the liver., (Copyright © 2020 Safarikia, Carpino, Overi, Cardinale, Venere, Franchitto, Onori, Alvaro and Gaudio.)

Background and Aims: Lipopolysaccharides (LPS) is increased in nonalcoholic fatty liver disease (NAFLD), but its relationship with liver inflammation is not defined., Approach and Results: We studied Escherichia coli LPS in patients with biopsy-proven NAFLD, 25 simple steatosis (nonalcoholic fatty liver) and 25 nonalcoholic steatohepatitis (NASH), and in mice with diet-induced NASH. NASH patients had higher serum LPS and hepatocytes LPS localization than controls, which was correlated with serum zonulin and phosphorylated nuclear factor-κB expression. Toll-like receptor 4 positive (TLR4 + ) macrophages were higher in NASH than simple steatosis or controls and correlated with serum LPS. NASH biopsies showed a higher CD61 + platelets, and most of them were TLR4 + . TLR4 + platelets correlated with serum LPS values. In mice with NASH, LPS serum levels and LPS hepatocyte localization were increased compared with control mice and associated with nuclear factor-κB activation. Mice on aspirin developed lower fibrosis and extent compared with untreated ones. Treatment with TLR4 inhibitor resulted in lower liver inflammation in mice with NASH., Conclusions: In NAFLD, Escherichia coli LPS may increase liver damage by inducing macrophage and platelet activation through the TLR4 pathway., (© 2020 by the American Association for the Study of Liver Diseases.)

Background and Aims: Mechanisms underlying the repair of extrahepatic biliary tree (EHBT) after injury have been scarcely explored. The aims of this study were to evaluate, by using a lineage tracing approach, the contribution of peribiliary gland (PBG) niche in the regeneration of EHBT after damage and to evaluate, in vivo and in vitro, the signaling pathways involved., Approach and Results: Bile duct injury was induced by the administration of 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 14 days to Krt19 Cre TdTomato LSL mice. Human biliary tree stem/progenitor cells (BTSC) within PBGs were isolated from EHBT obtained from liver donors. Hepatic duct samples (n = 10) were obtained from patients affected by primary sclerosing cholangitis (PSC). Samples were analyzed by histology, immunohistochemistry, western blotting, and polymerase chain reaction. DDC administration causes hyperplasia of PBGs and periductal fibrosis in EHBT. A PBG cell population (Cytokeratin19 - /SOX9 + ) is involved in the renewal of surface epithelium in injured EHBT. The Wnt signaling pathway triggers human BTSC proliferation in vitro and influences PBG hyperplasia in vivo in the DDC-mediated mouse biliary injury model. The Notch signaling pathway activation induces BTSC differentiation in vitro toward mature cholangiocytes and is associated with PBG activation in the DDC model. In human PSC, inflammatory and stromal cells trigger PBG activation through the up-regulation of the Wnt and Notch signaling pathways., Conclusions: We demonstrated the involvement of PBG cells in regenerating the injured biliary epithelium and identified the signaling pathways driving BTSC activation. These results could have relevant implications on the pathophysiology and treatment of cholangiopathies., (© 2019 by the American Association for the Study of Liver Diseases.)

Human biliary tree stem/progenitor cells (hBTSCs), reside in peribiliary glands, are mainly stimulated by primary sclerosing cholangitis (PSC) and cholangiocarcinoma. In these pathologies, hBTSCs displayed epithelial-to-mesenchymal transition (EMT), senescence characteristics, and impaired differentiation. Here, we investigated the effects of cholest-4,6-dien-3-one, an oxysterol involved in cholangiopathies, on hBTSCs biology. hBTSCs were isolated from donor organs, cultured in self-renewal control conditions, differentiated in mature cholangiocytes by specifically tailored medium, or exposed for 10 days to concentration of cholest-4,6-dien-3-one (0.14 mM). Viability, proliferation, senescence, EMT genes expression, telomerase activity, interleukin 6 (IL6) secretion, differentiation capacity, and HDAC6 gene expression were analyzed. Although the effect of cholest-4,6-dien-3-one was not detected on hBTSCs viability, we found a significant increase in cell proliferation, senescence, and IL6 secretion. Interestingly, cholest-4.6-dien-3-one impaired differentiation in mature cholangiocytes and, simultaneously, induced the EMT markers, significantly reduced the telomerase activity, and induced HDAC6 gene expression. Moreover, cholest-4,6-dien-3-one enhanced bone morphogenic protein 4 (Bmp-4) and sonic hedgehog (Shh) pathways in hBTSCs. The same pathways activated by human recombinant proteins induced the expression of EMT markers in hBTSCs. In conclusion, we demonstrated that chronic exposition of cholest-4,6-dien-3-one induced cell proliferation, EMT markers, and senescence in hBTSC, and also impaired the differentiation in mature cholangiocytes.

During foetal life, the liver plays the important roles of connection and transient hematopoietic function. Foetal liver cells develop in an environment called a hematopoietic stem cell niche composed of several cell types, where stem cells can proliferate and give rise to mature blood cells. Embryologically, at about the third week of gestation, the liver appears, and it grows rapidly from the fifth to 10th week under WNT/β-Catenin signaling pathway stimulation, which induces hepatic progenitor cells proliferation and differentiation into hepatocytes. Development of new strategies and identification of new cell sources should represent the main aim in liver regenerative medicine and cell therapy. Cells isolated from organs with endodermal origin, like the liver, bile ducts, and pancreas, could be preferable cell sources. Furthermore, stem cells isolated from these organs could be more susceptible to differentiate into mature liver cells after transplantation with respect to stem cells isolated from organs or tissues with a different embryological origin. The foetal liver possesses unique features given the co-existence of cells having endodermal and mesenchymal origin, and it could be highly available source candidate for regenerative medicine in both the liver and pancreas. Taking into account these advantages, the foetal liver can be the highest potential and available cell source for cell therapy regarding liver diseases and diabetes., Competing Interests: The authors declare no conflict of interest.

Cell adhesion is essential for survival, it plays important roles in physiological cell functions, and it is an innovative target in regenerative medicine. Among the molecular interactions and the pathways triggered during cell adhesion, the binding of cluster of differentiation 44 (CD44), a cell-surface glycoprotein involved in cell-cell interactions, to hyaluronic acid (HA), a major component of the extracellular matrix, is a crucial step. Cell therapy has emerged as a promising treatment for advanced liver diseases; however, so far, it has led to low cell engraftment and limited cell repopulation of the target tissue. Currently, different strategies are under investigation to improve cell grafting in the liver, including the use of organic and inorganic biomatrices that mimic the microenvironment of the extracellular matrix. Hyaluronans, major components of stem cell niches, are attractive candidates for coating stem cells since they improve viability, proliferation, and engraftment in damaged livers. In this review, we will discuss the new strategies that have been adopted to improve cell grafting and track cells after transplantation.

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota's Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies that may develop at any level of the biliary tree. CCA is currently classified into intrahepatic (iCCA), perihilar (pCCA) and distal (dCCA) on the basis of its anatomical location. Notably, although these three CCA subtypes have common features, they also have important inter- and intra-tumor differences that can affect their pathogenesis and outcome. A unique feature of CCA is that it manifests in the hepatic parenchyma or large intrahepatic and extrahepatic bile ducts, furnished by two distinct stem cell niches: the canals of Hering and the peribiliary glands, respectively. The complexity of CCA pathogenesis highlights the need for a multidisciplinary, translational, and systemic approach to this malignancy. This review focuses on advances in the knowledge of CCA histomorphology, risk factors, molecular pathogenesis, and subsets of CCA., Competing Interests: Conflict of Interest: None