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In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction., (© 2024. The Author(s).)

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.)

This study investigated the effects of estrogen and estrogen receptor alpha (ERα) on the pathogenesis of primary biliary cholangitis (PBC) in human intrahepatic bile duct epithelial cells (HiBECs). The researchers measured serum levels of ERα, oxidative stress indicators, and cytokines in PBC patients and healthy controls. They examined the expression of ERα, pyruvate dehydrogenase complex E2-component (PDC-E2), and apoptosis-related proteins in the small bile ducts. In vitro experiments with HiBECs showed that estrogen had a dual effect on cell viability, increasing it at low concentrations but reducing it at higher concentrations. ERα activation led to mitochondrial damage, apoptosis, and upregulation of ERα and PDC-E2 expression. These findings suggest that the high expression of ERα in the bile ducts contributes to mitochondrial damage, inflammation, and apoptosis in PBC. The study highlights ERα as a potential target for understanding and treating estrogen-mediated PBC pathogenesis., (© 2023 Wiley Periodicals LLC.)

Despite the robust healing capacity of the liver, regenerative failure underlies numerous hepatic diseases, including the JAG1 haploinsufficient disorder, Alagille syndrome (ALGS). Cholestasis due to intrahepatic duct (IHD) paucity resolves in certain ALGS cases but fails in most with no clear mechanisms or therapeutic interventions. We find that modulating jag1b and jag2b allele dosage is sufficient to stratify these distinct outcomes, which can be either exacerbated or rescued with genetic manipulation of Notch signaling, demonstrating that perturbations of Jag/Notch signaling may be causal for the spectrum of ALGS liver severities. Although regenerating IHD cells proliferate, they remain clustered in mutants that fail to recover due to a blunted elevation of Notch signaling in the distal-most IHD cells. Increased Notch signaling is required for regenerating IHD cells to branch and segregate into the peripheral region of the growing liver, where biliary paucity is commonly observed in ALGS. Mosaic loss- and-gain-of-function analysis reveals Sox9b to be a key Notch transcriptional effector required cell autonomously to regulate these cellular dynamics during IHD regeneration. Treatment with a small-molecule putative Notch agonist stimulates Sox9 expression in ALGS patient fibroblasts and enhances hepatic sox9b expression, rescues IHD paucity and cholestasis, and increases survival in zebrafish mutants, thereby providing a proof-of-concept therapeutic avenue for this disorder.

Aims: This study aimed to explore the protection mechanism of ISO-1 on severe acute pancreatitis-associated intrahepatic bile duct (IBD) injury in rats., Methods: Forty-eight specific-pathogen-free male Wistar rats were randomly divided into four groups (N = 12): a sham operation group (SO group), a severe acute pancreatitis model group (SAP group), a ISO-1 treatment group (ISO-1 + SAP group), and a ISO-1 control group (ISO-1 + SO group). All rats were killed after 12 h of being made models. Immunohistochemistry was used to detect the expression of MIF and P38 in IBD cells. MIF mRNA expression in IBD cells was observed using real-time fluorescent quantitative polymerase chain reaction (real-time PCR). In addition, Western blotting was performed to detect the protein expression of P38, phosphorylated P38 (P-P38), nuclear factor-κB (NF-κB p65), and tumor necrosis factor alpha (TNF-α). Enzyme-linked immunosorbent assays were used to analyze the levels of TNF-α, IL-1β, and IL-6 in the IBD of rats., Results: Compared with SAP, after treatment with ISO-1, the pathological injuries of pancreas, liver, and IBD cells in ISO-1 treatment group remarkably relieved. The expression of MIF in the IBD cells was significantly downregulated both at mRNA and at protein levels in ISO-1 treatment group. Besides, the protein expression levels of P38, P-P38, NF-κBp65, TNF-α, IL-1β, and IL-6 in the IBD in rats were also significantly decreased in ISO-1 treatment group (all P < 0.05)., Conclusion: ISO-1 may protect the IBD cells, reduce pathological injuries, and reduce the inflammatory response in SAP rats. Its mechanisms may be via inhibiting the expression of MIF and then blocking the activation of p38-MAPK and NF-κB signaling pathway., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

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.)

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Biliary tract cancer (BTC) arises from biliary epithelial cells (BECs) and includes intrahepatic cholangiocarcinoma (IHCC), gallbladder cancer (GC), and extrahepatic cholangiocarcinoma (EHCC). Although frequent KRAS mutations and epigenetic changes at the INK4A/ARF locus have been identified, the molecular pathogenesis of BTC is unclear and the development of corresponding anticancer agents remains inadequate. We isolated epithelial cell adhesion molecule (EpCAM)-positive BECs from the mouse intrahepatic bile duct, gallbladder, and extrahepatic bile duct, and established organoids derived from these cells. Introduction of activated KRAS and homozygous deletion of Ink4a/Arf in the cells of each organoid type conferred the ability to form lethal metastatic adenocarcinoma with differentiated components and a pronounced desmoplastic reaction on cell transplantation into syngeneic mice, indicating that the manipulated cells correspond to BTC-initiating cells. The syngeneic mouse models recapitulate the pathological features of human IHCC, GC, and EHCC, and they should therefore prove useful for the investigation of BTC carcinogenesis and the development of new therapeutic strategies. Tumor cells isolated from primary tumors formed organoids in three-dimensional culture, and serial syngeneic transplantation of these cells revealed that their cancer stem cell properties were supported by organoid culture, but not by adherent culture. Adherent culture thus attenuated tumorigenic activity as well as the expression of both epithelial and stem cell markers, whereas the expression of epithelial-mesenchymal transition (EMT)-related transcription factor genes and mesenchymal cell markers was induced. Our data show that organoid culture is important for maintenance of epithelial cell characteristics, stemness, and tumorigenic activity of BTC-initiating cells., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background & Aims: Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9 EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types., Methods: Sox9 EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9 EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence., Results: BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9 EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP high BECs. Single Sox9 EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9 EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation., Conclusions: Our data demonstrate that Sox9 EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Organoids provide a powerful system to study epithelia in vitro. Recently, this approach was applied successfully to the biliary tree, a series of ductular tissues responsible for the drainage of bile and pancreatic secretions. More precisely, organoids have been derived from ductal tissue located outside (extrahepatic bile ducts; EHBDs) or inside the liver (intrahepatic bile ducts; IHBDs). These organoids share many characteristics, including expression of cholangiocyte markers such as keratin (KRT) 19. However, the relationship between these organoids and their tissues of origin, and to each other, is largely unknown., Approach and Results: Organoids were derived from human gallbladder, common bile duct, pancreatic duct, and IHBDs using culture conditions promoting WNT signaling. The resulting IHBD and EHBD organoids expressed stem/progenitor markers leucine-rich repeat-containing G-protein-coupled receptor 5/prominin 1 and ductal markers KRT19/KRT7. However, RNA sequencing revealed that organoids conserve only a limited number of regional-specific markers corresponding to their location of origin. Of particular interest, down-regulation of biliary markers and up-regulation of cell-cycle genes were observed in organoids. IHBD and EHBD organoids diverged in their response to WNT signaling, and only IHBDs were able to express a low level of hepatocyte markers under differentiation conditions., Conclusions: Taken together, our results demonstrate that differences exist not only between extrahepatic biliary organoids and their tissue of origin, but also between IHBD and EHBD organoids. This information may help to understand the tissue specificity of cholangiopathies and also to identify targets for therapeutic development., (© 2020 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Cholangiopathies are chronic, progressive diseases of the biliary tree, and can be either acquired or genetic. The primary target is the cholangiocyte (CC), the cell type lining the bile duct that is responsible for bile modification and transport. Despite advances in our understanding and diagnosis of these diseases in recent years, there are no proven therapeutic treatments for the majority of the cholangiopathies, and liver transplantation is the only life-extending treatment option for patients with end-stage cholestatic liver disease. One potential therapeutic strategy is to facilitate endogenous repair of the biliary system, which may alleviate intrahepatic cholestasis caused by these diseases. During biliary injury, hepatocytes (HC) are known to alter their phenotype and acquire CC-like features, a process known as cellular reprogramming. This brief review discusses the potential ways in which reprogrammed HC may contribute to biliary repair, thereby restoring bile flow and reducing the severity of cholangiopathies. Some of these include modifying bile to reduce toxicity, serving as a source of de novo CC to repair the biliary epithelium, or creating new channels to facilitate bile flow.

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks were lost. The canalicular collapse was an incipient defect commonly associated with hepatocyte injury irrespective of cholestatic statuses, and could sufficiently provoke the ductular reaction when artificially induced. We propose a unifying model for the induction of the ductular reaction, where compensatory biliary epithelial tissue remodeling ensures bile-excreting network homeostasis.

Aberrant Notch and Wnt signaling are known drivers of cholangiocarcinoma (CCA), but the underlying factors that initiate and maintain these pathways are not known. Here, we show that the proline-rich homeodomain protein/hematopoietically expressed homeobox (PRH/HHEX) transcription factor forms a positive transcriptional feedback loop with Notch3 that is critical in CCA. PRH/HHEX expression is elevated in CCA, and depletion of PRH reduces CCA tumor growth in a xenograft model. Overexpression of PRH in primary human biliary epithelial cells is sufficient to increase cell proliferation and produce an invasive phenotype. Interrogation of the gene networks regulated by PRH and Notch3 reveals that unlike Notch3, PRH directly activates canonical Wnt signaling. These data indicate that hyperactivation of Notch and Wnt signaling is independent of the underlying mutational landscape and has a common origin in dysregulation of PRH. Moreover, they suggest new therapeutic options based on the dependence of specific Wnt, Notch, and CDK4/6 inhibitors on PRH activity. SIGNIFICANCE: The PRH/HHEX transcription factor is an oncogenic driver in cholangiocarcinoma that confers sensitivity to CDK4/6 inhibitors., (©2019 American Association for Cancer Research.)

Background: Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by the highly selective autoimmune injury of small intrahepatic bile ducts. Studies reported that the cholangiocytes from PBC patients expressed significantly higher levels of both receptor activator of nuclear factor-kappa B (RANK) and its ligand RANKL. However, the accurate role of RANK/RANKL axis in PBC remains unclear., Methods: Forty patients with PBC were enrolled according to the inclusion criteria. The biochemical parameters (alkaline phosphatase, ALP; gamma-glutamyltransferase, GGT; alanine aminotransferase, ALT; aspartate transaminase, AST; total bilirubin, TB) were collected at baseline and followed-up after 6 months of treatment with ursodeoxycholic acid (UDCA, 15 mg/kg d). Stages of PBC were diagnosed based on liver biopsy histopathology according to Nakanuma's criteria. RANK expression in hepatic tissues was detected by immunohistochemistry. The cellular immunofluorescence method was used to locate the distribution of RANK in the human intrahepatic biliary epithelial cells (HIBECs) cultured in vitro. HIBECs were treated with RANKL at a concentration of 100 ng/ml or transfected with RANK-overexpressing lentivirus (LV-RANK). CCK-8 assay and cell cycle assay were used to detect the cell proliferation. Real-time PCR was used to detect the expression of IL-6, E-cadherin, VCAM, ICAM-1, TNF-α, and CD80., Results: RANK expression in liver biopsies from early PBC patients (stage I + stage II) was significantly lower than that from advanced PBC patients (stage III + stage IV) (1.7 ± 0.63 vs. 2.3 ± 0.45 scores, P < 0.05). High-RANK patients seemed to have better response to UDCA than low-RANK patients (88.9% vs. 40.9%, P < 0.05). The baseline biochemical parameters between the two groups were comparable. The decline percentages of ALP and GGT after UDCA treatment were more obvious in high-RANK patients than those in low-RANK patients (53.90% ± 9.82% vs. 23.93% ± 6.24%, P < 0.05; 74.11% ± 7.18% vs. 48.00% ± 8.17%, P < 0.05, respectively). HIBECs proliferation was significantly inhibited after treatment of RANKL or transfection with LV-RANK. Increased expression of IL-6 and E-cadherin was observed in HIBECs treated with RANKL or LV-RANK., Conclusion: The overall hepatic RANK expression was associated with disease severity and biochemical response in PBC patients. Activation of RANK/RANKL signaling pathway inhibited cholangiocytes proliferation in vitro. Our study suggested that RANK/RANKL pathway might be a potential target of immunotherapy of PBC based on its involvement in the occurrence and development of the disease.

Cholangiocarcinoma (CCA) is a lethal disease with increasing incidence worldwide. Previous study showed that CCA was sensitive to adenosine. Thereby, molecular mechanisms of CCA inhibition by adenosine were examined in this study. Our results showed that adenosine inhibited CCA cells via an uptake of adenosine through equilibrative nucleoside transporters (ENTs), instead of activation of adenosine receptors. The inhibition of ENTs by NBTI caused the inhibitory effect of adenosine to subside, while adenosine receptor antagonists, caffeine and CGS-15943, failed to do so. Intracellular adenosine level was increased after adenosine treatment. Also, a conversion of adenosine to AMP by adenosine kinase is required in this inhibition. On the other hand, inosine, which is a metabolic product of adenosine has very little inhibitory effect on CCA cells. This indicates that a conversion of adenosine to inosine may reduce adenosine inhibitory effect. Furthermore, there was no specific correlation between level of proinflammatory proteins and CCA responses to adenosine. A metabolic stable analog of adenosine, 2Cl-adenosine, exerted higher inhibition on CCA cell growth. The disturbance in intracellular AMP level also led to an activation of 5′ AMP-activated protein kinase (AMPK). Accordingly, we proposed a novel adenosine-mediated cancer cell growth and invasion suppression via a receptor-independent mechanism in CCA.

Cholangiocarcinoma (CCA) represents a type of epithelial cancer with a late diagnosis and poor outcome. However, the molecular mechanisms responsible for the development of CCA have not yet been fully identified. Thus, in this study, we aimed to elucidate some of these mechanisms. For this purpose, isobaric tags for relative and absolute quantification (iTRAQ) was performed to analyze the secretory proteins from the 2 CCA cell lines, TFK1 and HuCCT1, as well as from a normal biliary epithelial cell line, human intrahepatic biliary epithelial cells (HiBECs). Differentially expressed proteins (DEPs) were identified and biological process analysis was performed according to the Gene Ontology (GO) functional classification annotation and KEGG metabolic pathway map analysis. tumor protein D52 (TPD52) and DnaJ heat shock protein family (Hsp40) member B1 (DNAJB1) were validated using RT‑qPCR, western blot analysis and immunohistochemistry. In total, 778 proteins were identified as DEPs. Following validation, TPD52 and DNAJB1 were used for further analysis. The expression levels of TPD52 and DNAJB1 were elevated in CCA cell lines, tissues and bile samples, suggesting that these proteins may contribute to tumor pathogenesis. In addition, the expression levels of TPD52 and DNAJB1 were found to be closely associated with the clinical parameters and prognosis of patients with CCA. On the whole, the findings of this study indicate that TPD52 and DNAJB1 may serve as novel bile biomarkers for CCA.

The liver is a complex organ that performs several functions to maintain homeostasis. These functions are modulated by calcium, a second messenger that regulates several intracellular events. In hepatocytes and cholangiocytes, which are the epithelial cell types in the liver, inositol 1,4,5-trisphosphate (InsP 3 ) receptors (ITPR) are the only intracellular calcium release channels. Three isoforms of the ITPR have been described, named type 1, type 2 and type 3. These ITPR isoforms are differentially expressed in liver cells where they regulate distinct physiological functions. Changes in the expression level of these receptors correlate with several liver diseases and hepatic dysfunctions. In this review, we highlight how the expression level, modulation, and localization of ITPR isoforms in hepatocytes and cholangiocytes play a role in hepatic homeostasis and liver pathology., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest. No financial support., (©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.)

Gradual alterations of cell's physiology and functions due to age or exposure to various stresses lead to the conversion of normal cells to senescent cells. Once becoming senescent, the cell stops dividing permanently but remains metabolically active. Cellular senescence does not have a single marker but is characterized mainly by a combination of multiple markers, such as, morphological changes, expression of cell cycle inhibitors, senescence associated β-galactosidase activity, and changes in nuclear membrane. When cells in an organ become senescent, the entire organism can be affected. This may occur through the senescence-associated secretory phenotype (SASP). SASP may exert beneficial or harmful effects on the microenvironment of tissues. Research on senescence has become a very exciting field in cell biology since the link between age-related diseases, including cancer, and senescence has been established. The loss of regenerative and homeostatic capacity of the liver over the age is somehow connected to cellular senescence. The major contributors of senescence properties in the liver are hepatocytes and cholangiocytes. Senescent cells in the liver have been implicated in the etiology of chronic liver diseases including cirrhosis and hepatocellular carcinoma and in the interference of liver regeneration. This review summarizes recently reported findings in the understanding of the molecular mechanisms of senescence and its relationship with liver diseases., Competing Interests: Conflict-of-interest statement: The authors have declared that no conflict of interest exists.

Objectives While bile duct brush cytology during endoscopic retrograde cholangiopancreatography (ERCP) is a well-established procedure for detecting malignant biliary stricture, its sensitivity is reportedly low. We aimed to determine the pre-ERCP factors affecting brush cytology sensitivity. Methods We retrospectively analyzed 185 patients who underwent brush cytology during the first ERCP for undiagnosed biliary stricture at our institution between January 2014 and December 2016. We analyzed the relationship of age, sex, final diagnosis, stricture location, tumor size, stricture length, total bilirubin level, white blood cell count, and C-reactive protein level with brush cytology sensitivity. Results The following conditions were established as final diagnoses: benign disease, 19 cases (10.3%); intrahepatic cholangiocarcinoma, 10 cases (5.4%); hilar cholangiocarcinoma, 38 cases (20.5%); extrahepatic cholangiocarcinoma, 44 cases (23.8%); pancreatic cancer, 55 cases (29.7%); other malignant tumors, 19 cases (10.3%). The sensitivity and specificity of brush cytology were 60.8% and 94.7%, respectively. The stricture length, total bilirubin level, and white blood cell count in true-positive cases were significantly higher than those in false-negative cases. Furthermore, a stratified analysis of the bilirubin levels demonstrated that sensitivity was highest in patients with moderate jaundice (80% for a total bilirubin level of 10-20 mg/dL), but significantly lower in patients with severe jaundice (total bilirubin level ≥20 mg/dL). Conclusion While the sensitivity of brush cytology increases with bilirubin levels of up to 20 mg/dL, severe jaundice has a negative effect on sensitivity, warranting additional pathological examinations according to the pre-ERCP bilirubin level.

The essential liver exocrine and endocrine functions require a precise spatial arrangement of the hepatic lobule consisting of the central vein, portal vein, hepatic artery, intrahepatic bile duct system, and hepatocyte zonation. This allows blood to be carried through the liver parenchyma sampled by all hepatocytes and bile produced by the hepatocytes to be carried out of the liver through the intrahepatic bile duct system composed of cholangiocytes. The molecular orchestration of multiple signaling pathways and epigenetic factors is required to set up lineage restriction of the bipotential hepatoblast progenitor into the hepatocyte and cholangiocyte cell lineages, and to further refine cell fate heterogeneity within each cell lineage reflected in the functional heterogeneity of hepatocytes and cholangiocytes. In addition to the complex molecular regulation, there is a complicated morphogenetic choreography observed in building the refined hepatic epithelial architecture. Given the multifaceted molecular and cellular regulation, it is not surprising that impairment of any of these processes can result in acute and chronic hepatobiliary diseases. To enlighten the development of potential molecular and cellular targets for therapeutic options, an understanding of how the intricate hepatic molecular and cellular interactions are regulated is imperative. Here, we review the signaling pathways and epigenetic factors regulating hepatic cell lineages, fates, and epithelial architecture., (© 2019 Elsevier Inc. All rights reserved.)

Incorporation of bile drainage system into engineered liver tissue is an important issue to advance liver regenerative medicine. Our group reported that three-dimensional (3D) coculture of fetal liver cells (FLCs) and adult rat biliary epithelial cells (BECs) allows reconstruction of hepatic spheroids that possess bile ductular structures. In this chapter, we describe the detailed protocol to isolate FLCs and BECs and to generate the spheroids with bile drainage system using these two types of primary cells.

Background and Aim: Intrahepatic biliary epithelial cells (IBECs) of the bile duct in liver tissue of patients with hepatolithiasis promoted the development of diseases through epithelial-mesenchymal transition (EMT). This study investigated whether lipopolysaccharide (LPS), a cell-wall constituent of gram-negative bacteria, could induce EMT of IBECs and toll-like receptor 4 (TLR4) had a regulatory role via activating the nuclear factor-κB (NF-κB)/Snail signaling pathway during this process in vivo., Methods: TLR4 short hairpin RNA (shRNA) adenovirus or negative control shRNA (NC shRNA) adenovirus (1 × 10 9 plaque-forming unit (PFU), respectively) was injected into the caudal vein of rats. After 96 h, 1 mg/kg LPS was infused retrogradely into the common bile duct for 48 h per rat. The effects of TLR4 shRNA on LPS-induced EMT were determined by evaluating the histopathological changes in IBECs using hematoxylin and eosin staining and the changes in the levels of EMT markers, TLR4, NF-κB p65, pNF-κB p65, and Snail using real-time polymerase chain reaction and Western blot analysis., Results: Compared with normal saline treatment, a loss of epithelial cell markers (E-cadherin and cytokeratin 7) and a gain of mesenchymal cell markers (N-cadherin and matrix metalloproteinase 2) were revealed. The levels of TLR4, NF-κB phosphorylation, and Snail significantly increased after LPS treatment, whereas pretreatment with TLR4 shRNA inhibited the LPS-induced EMT by downregulating the NF-κB/Snail signaling pathway., Conclusions: LPS induced the EMT of IBECs by activating TLR4. The RNAi-mediated knockdown of TLR4 suppressed EMT occurrence via downregulating the NF-κB/Snail signaling pathway, implicating TLR4 as a new target for human hepatolithiasis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Cholangiocarcinoma (CCA), the cancer arising from the epithelial cells of bile ducts, is a prototype of inflammatory-driven cancer. Cytokines released by cancer associated fibroblasts (CAFs) play a pivotal role in CCA progression, driving the epigenetic Epithelial-to-Mesenchymal transition and the growth and metastasization of CCA cells. Consistently, the conditioned medium from CCA-derived CAFs further stimulated the secretion of IL-6, and to a lesser extent of IL-8, by CCA cells. CCA has a poor prognosis, because of late diagnosis and of high resistance to radio- and chemo-therapy of CCA cells. Targeting the CAFs and their secretion could be an alternative option. We found that while IL-6 indeed promoted the cell migration of invasive CCA cells, the nutraceutical Resveratrol strongly counteracted this effect both in CCA cells and in immortalized cholangiocytes. More importantly, here we show that Resveratrol has the potential to abrogate the secretion of IL-6 by CAFs. While the conditioned medium from CAFs strongly induced IL-6 mediated motility of CCA cells, the conditioned medium from CAFs pre-treated with Resveratrol completely halted cancer cell motility and reverted the N-to E-cadherin switch in migrating cells. This effect was associated with stimulation of autophagy in the cancer cells. This is the first demonstration that CAFs secretory products directly affect the regulation of autophagy and consequently the behavior of CCA cells, and that a nutraceutical may revert the malignant phenotype of cancer cells by acting on CAFs metabolism and secretion., (Copyright © 2018. Published by Elsevier B.V.)

The initiation and progression of liver cancer, including hepatocellular carcinoma and intrahepatic cholangiocarcinoma, are dependent on its tumor microenvironment. Immune cells are key players in the liver cancer microenvironment and show complicated crosstalk with cancer cells. Emerging evidence has shown that the functions of immune cells are closely related to cell metabolism. However, the effects of metabolic changes of immune cells on liver cancer progression are largely undefined. In this review, we summarize the recent findings of immunometabolism and relate these findings to liver cancer progression. We also explore the translation of the understanding of immunometabolism for clinical use., Competing Interests: Conflict-of-interest statement: The authors declare that they have no competing interests.

The biliary tree is an essential component of transplantable human liver tissue. Despite recent advances in liver tissue engineering, attempts at re-creating the intrahepatic biliary tree have not progressed significantly. The finer branches of the biliary tree are structurally and functionally complex and heterogeneous and require harnessing innate developmental processes for their regrowth. Here we demonstrate the ability of decellularized liver extracellular matrix (dECM) hydrogels to induce the in vitro formation of complex biliary networks using encapsulated immortalized mouse small biliary epithelial cells (cholangiocytes). This phenomenon is not observed using immortalized mouse large cholangiocytes, or with purified collagen 1 gels or Matrigel. We also show phenotypic stability via immunostaining for specific cholangiocyte markers. Moreover, tight junction formation and maturation was observed to occur between cholangiocytes, exhibiting polarization and transporter activity. To better define the mechanism of duct formation, we utilized three fluorescently labeled, but otherwise identical populations of cholangiocytes. The cells, in a proximity dependent manner, either branch out clonally, radiating from a single nucleation point, or assemble into multi-colored structures arising from separate populations. These findings present liver dECM as a promising biomaterial for intrahepatic bile duct tissue engineering and as a tool to study duct remodeling in vitro.

Biliary epithelial cells (BEC) are closely related to some immune regulatory bile duct diseases. However, the complexity and polymorphism of the morphology and function of bile duct cells have hindered further investigation. Therefore, the aim of this study is to investigate how interleukin-6 (IL-6) affects the migration, cellular senescence, and apoptosis of human intrahepatic biliary epithelial cells (HIBECs). The HIBECs were stimulated by different concentrations of IL-6 (0, 5, 10, 15, and 20 ng/mL, respectively). Transwell assay was performed in order to measure the migration abilities, positive β-Galactosidase staining for the cellular senescence of HIBECs, MTT assay for changes of proliferation after IL-6 treatment and flow cytometry for cell cycle and apoptosis. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting were conducted in order to detect the mRNA and protein expressions of epithelial-mesenchymal transition (EMT) markers in HIBECs. In comparison to the 0 ng/mL group, in the 5, 10, 15, and 20 ng/mL groups, a significant increase in the number of migratory HIBECs, proliferation, along with mRNA and protein expressions of EMT markers was observed. While the mRNA and protein expressions of epithelial markers, the number of β-galactosidase positive staining cells, as well as apoptosis rate of HIBECs dramatic decreased. Further, the aforementioned changes were significantly more evident in the 15 and 20 ng/mL groups in comparison to the 5 and 10 ng/mL groups. IL-6 may stimulate EMT, enhance the migration and proliferation, and inhibit apoptosis of HIBECs, thus delaying cellular senescence., (© 2017 Wiley Periodicals, Inc.)

The biliary system plays an important role in several acquired and genetic disorders of the liver. We have previously shown that biliary duct epithelium contains cells giving rise to proliferative Lgr5 + organoids in vitro. However, it remained unknown whether all biliary cells or only a specific subset had this clonogenic activity. The cell surface protease ST14 was identified as a positive marker for the clonogenic subset of cholangiocytes and was used to separate clonogenic and non-clonogenic duct cells by fluorescence-activated cell sorting. Only ST14 hi duct cells had the ability to generate organoids that could be serially passaged. The gene expression profiles of clonogenic and non-clonogenic duct cells were similar, but several hundred genes were differentially expressed. RNA fluorescence in situ hybridization showed that clonogenic duct cells are interspersed among regular biliary epithelium at a ∼1:3 ratio. We conclude that adult murine cholangiocytes can be subdivided into two populations differing in their proliferative capacity., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

After liver injury, regeneration occurs through self-replication of hepatocytes. In severe liver injury, hepatocyte proliferation is impaired-a feature of human chronic liver disease. It is unclear whether other liver cell types can regenerate hepatocytes. Here we use two independent systems to impair hepatocyte proliferation during liver injury to evaluate the contribution of non-hepatocytes to parenchymal regeneration. First, loss of β1-integrin in hepatocytes with liver injury triggered a ductular reaction of cholangiocyte origin, with approximately 25% of hepatocytes being derived from a non-hepatocyte origin. Second, cholangiocytes were lineage traced with concurrent inhibition of hepatocyte proliferation by β1-integrin knockdown or p21 overexpression, resulting in the significant emergence of cholangiocyte-derived hepatocytes. We describe a model of combined liver injury and inhibition of hepatocyte proliferation that causes physiologically significant levels of regeneration of functional hepatocytes from biliary cells.

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A -/- mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4 + lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c + dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6C hi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A + cells was positively correlated with the degree of CD68 + macrophage infiltration at diagnosis. Hepatic CD4 + lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation., Conclusion: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188)., (© 2016 by the American Association for the Study of Liver Diseases.)

This study aimed to investigate the function of Activatable Cell-Penetrating Peptides (ACPP) in detecting the changes of human intrahepatic bile duct epithelial cell(hIBDEC). ACPP, which target matrix metalloproteinases, were constructed. All were labeled with FITC and Gd-DTPA at the N-terminal. Fluorescence microscopy was used to observe the fluorescence intensity inside hIBDEC after stimulating with different concentrations of LPS and incubating with different concentrations of ACPP to determine the optimal concentration range for LPS stimulation and the optimal concentration for FITC-ACPP effect. Flow cytometry and magnetic resonance imaging were used to detect fluorescence signal intensity and nuclear magnetic resonance signal intensity, respectively, after stimulating with different concentrations of LPS. LPS stimulation time and ACPP incubation time were also evaluated, and variance analysis was conducted to analyze intracellular signal change characteristics for every group. Activatable Cell-Penetrating Peptides (ACPP), which were marked with FITC and Gd-DTPA had target-penetrating activity. The intracellular signal intensity gradually increased with the increase in LPS stimulation time and ACPP incubation time within a certain range; however, it did not increase with the increase of LPS concentration. ACPP can be used for imaging hIBDEC with epithelial-mesenchymal transition.

Unlabelled: The intrahepatic bile duct (IHBD) is a highly organized tubular structure consisting of cholangiocytes, biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Although several models have been proposed, it remains unclear how the three-dimensional (3D) IHBD network develops during liver organogenesis. Using 3D imaging techniques, we demonstrate that the continuous luminal network of IHBDs is established by 1 week after birth. Beyond this stage, the IHBD network consists of large ducts running along portal veins (PVs) and small ductules forming a mesh-like network around PVs. By analyzing embryonic and neonatal livers, we found that newly differentiated cholangiocytes progressively form a continuous and homogeneous luminal network. Elongation of this continuous network toward the liver periphery was attenuated by a potent Notch-signaling inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. Subsequent to this first step, the fine homogenous network is reorganized into the mature hierarchical network consisting of large ducts and small ductules. Between E17 and E18, when the homogenous network is radically reorganized into the mature hierarchical network, bile canaliculi rapidly extend and bile flow into IHBDs may increase. When formation of bile canaliculi was blocked between E16 and E18 by a multidrug resistance protein 2 inhibitor (benzbromarone), the structural rearrangement of IHBDs was significantly suppressed., Conclusion: Establishment of the mature IHBD network consists of two sequential events: (1) formation of the continuous luminal network regulated by the Notch-signaling pathway and (2) dynamic rearrangement of the homogeneous network into the hierarchical network induced by increased bile flow resulting from the establishment of hepatobiliary connections. (Hepatology 2016;64:175-188)., (© 2016 by the American Association for the Study of Liver Diseases.)

Hepatic expression levels of CXCL12, a chemokine important in inflammatory and stem cell recruitment, and its receptor, C-X-C chemokine receptor 4, are increased during all forms of liver injury. CXCL12 is expressed by both parenchymal and nonparenchymal hepatic cells, and on the basis of immunohistochemistry, biliary epithelial cells (BECs) are thought to be a predominant source of hepatic CXCL12, thereby promoting periportal recruitment of C-X-C chemokine receptor 4-expressing lymphocytes. Our study aims to show that BECs may, in fact, not be the predominant source of hepatic CXCL12. We measured CXCL12 secretion and expression from human and murine BECs using enzyme-linked immunosorbent assay and Western blot analysis from cell culture supernatants and whole cell lysates, respectively, whereas CXCL12 expression in murine livers was analyzed in a Cxcl12-Gfp reporter mouse. Cell culture supernatants and whole cell lysates from BECs failed to demonstrate their expression of CXCL12. Furthermore, we confirmed these results with a Cxcl12-Gfp reporter mouse in which green fluorescent protein expression is notably absent from BECs. Interestingly, on the basis of green fluorescent protein expression, we demonstrate a population of CXCL12-expressing cells within the portal tract that are distinct, yet intimately associated with BECs. These findings indicate that BECs are not a predominant source of CXCL12., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Most cholestatic disorders are caused by defects in cholangiocytes. The type 3 isoform of the inositol 1,4,5-trisphosphate receptor (ITPR3) is the most abundant intracellular calcium release channel in cholangiocytes. ITPR3 is required for bicarbonate secretion by bile ducts, and its expression is reduced in intrahepatic bile ducts of patients with cholestatic disorders. We investigated whether the nuclear factor, erythroid 2-like 2 (NFE2L2 or NRF2), which is sensitive to oxidative stress, regulates expression of ITPR3., Methods: The activity of the ITPR3 promoter was measured in normal human cholangiocyte (NHC) cells and primary mouse cholangiocytes. Levels of ITPR3 protein and messenger RNA were examined by immunoblot and polymerase chain reaction analyses, respectively. ITPR3 activity was determined by measuring calcium signaling in normal human cholangiocyte cells and secretion in isolated bile duct units. Levels of NRF2 were measured in liver tissues from rats with cholestasis (induced by administration of α-napthylisothiocyanate) and from patients with biliary diseases., Results: We identified a musculo-aponeurotic fibrosarcoma recognition element in the promoter of ITPR3 that bound NRF2 directly in NHC cells and mouse cholangiocytes. Increasing binding of NRF2 at this site resulted in chromatin remodeling that reduced promoter activity. Mutant forms of the musculo-aponeurotic fibrosarcoma recognition element did not bind NRF2. Activation of NRF2 with quercetin or by oxidative stress reduced expression of ITPR3 and calcium signaling in NHC cells; quercetin also reduced secretion by bile duct units isolated from rats. Knockdown of NRF2 with small interfering RNAs restored expression and function of ITPR3 in NHC cells incubated with quercetin. Bile ducts from rats with cholestasis and patients with cholangiopathic disorders expressed higher levels of NRF2 and lower levels of ITPR3 than ducts from control rats or patients with other liver disorders., Conclusions: The transcription factor NRF2 binds to the promoter of ITPR3 to inhibit its expression in cholangiocytes, leading to reduced calcium signaling and bile duct secretion. This could be a mechanism by which oxidative stress inhibits these processes and contributes to cholangiopathies., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Serving as the center for metabolism and detoxification, the liver is inherently susceptible to a wide variety of damage imposed by toxins or chemicals. Induction of cell populations with biliary epithelial phenotypes, which include progenitor-like cells and are referred to as liver progenitor cells, is often observed in histopathological examination of various liver diseases in both human patients and animal models and has been implicated in regeneration. However, the tissue dynamics underlying this phenomenon remains largely unclear. We have developed a simple imaging technique to reveal the global and fine-scale architecture of the biliary tract spreading in the mouse liver. Using this novel method, we show that the emergence and expansion of liver progenitor cells actually reflect structural transformation of the intrahepatic biliary tree in mouse liver injury models. The biliary branches expanded their area gradually and contiguously along with the course of chronic injury. Relevant regulatory signals known to be involved in liver progenitor cell regulation, including fibroblast growth factor 7 and tumor necrosis factor-like weak inducer of apoptosis, can modulate the dynamics of the biliary epithelium in different ways. Importantly, the structural transformations of the biliary tree were diverse and corresponded well with the parenchymal injury patterns. That is, when chronic hepatocyte damage was induced in the pericentral area, the biliary branches exhibited an extended structure from the periportal area with apparent tropism toward the distant injured area., Conclusion: The hepatobiliary system possesses a unique and unprecedented structural flexibility and can remodel dynamically and adaptively in response to various injury conditions; this type of tissue plasticity should constitute an essential component to maintain liver homeostasis., (© 2015 by the American Association for the Study of Liver Diseases.)

There are currently no reports of identification of stem cells in human gallbladder. The differences between human gallbladder and intrahepatic bile duct (IHBD) cells have also not been explored. The goals of this study were to evaluate if human fetal gallbladder contains a candidate stem cell population and if fetal gallbladder cells are distinct from fetal IHBD cells. We found that EpCAM+CD44+CD13+ cells represent the cell population most enriched for clonal self-renewal from primary gallbladder. Primary EpCAM+CD44+CD13+ cells gave rise to EpCAM+CD44+CD13+ and EpCAM+CD44+CD13- cells in vitro, and gallbladder cells expanded in vitro exhibited short-term engraftment in vivo. Last, we found that CD13, CD227, CD66, CD26 and CD49b were differentially expressed between gallbladder and IHBD cells cultured in vitro indicating clear phenotypic differences between the two cell populations. Microarray analyses of expanded cultures confirmed that both cell types have unique transcriptional profiles with predicted functional differences in lipid, carbohydrate, nucleic acid and drug metabolism. In conclusion, we have isolated a distinct clonogenic population of epithelial cells from primary human fetal gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

During cholestatic liver disease, there is dysregulation in the balance between biliary growth and loss in bile duct-ligated (BDL) rats modulated by neuroendocrine peptides via autocrine/paracrine pathways. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone that modulates reproductive function and proliferation in many cell types. We evaluated the autocrine role of GnRH in the regulation of cholangiocyte proliferation. The expression of GnRH receptors was assessed in a normal mouse cholangiocyte cell line (NMC), sham, and BDL rats. The effect of GnRH administration was evaluated in normal rats and in NMC. GnRH-induced biliary proliferation was evaluated by changes in intrahepatic bile duct mass and the expression of proliferation and function markers. The expression and secretion of GnRH in NMC and isolated cholangiocytes was assessed. GnRH receptor subtypes GnRHR1 and GnRHR2 were expressed in cholangiocytes. Treatment with GnRH increased intrahepatic bile duct mass as well as proliferation and function markers in cholangiocytes. Transient knockdown and pharmacologic inhibition of GnRHR1 in NMC decreased proliferation. BDL cholangiocytes had increased expression of GnRH compared with normal rats, accompanied by increased GnRH secretion. In vivo and in vitro knockdown of GnRH decreased intrahepatic bile duct mass/cholangiocyte proliferation and fibrosis. GnRH secreted by cholangiocytes promotes biliary proliferation via an autocrine pathway. Disruption of GnRH/GnRHR signaling may be important for the management of cholestatic liver diseases., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Canalicular bile is secreted by hepatocytes and then passes through the intrahepatic bile ducts, comprised of cholangiocytes, to reach the extrahepatic biliary system. In addition to providing a conduit for bile to drain from the liver, cholangiocytes play an active role in modifying bile composition. Bile formation is the result of a series of highly coordinated intricate membrane-transport interactions. Proper systematic regulation of solute and water transport is critical for both digestion and the health of the liver, yet our knowledge of cholangiocyte water and ion transporters and their relative expression patterns remains incomplete. In this report, we provide a comprehensive expression profile of the aquaporin (AQP) family and three receptors/channels known to regulate ion transport in the murine cholangiocyte. In murine intrahepatic cholangiocytes, we found mRNA expression for all twelve of the members of the AQP family of proteins and found temporal changes in the expression profile occurring with age. Using AQP4, an established marker within cholangiocyte physiology, we found that AQP2, AQP5 and AQP6 expression levels to be significantly different between the neonatal and adult time points. Furthermore, there were distinct temporal expression patterns, with that of AQP12 unique in that its expression level decreased with age, whilst the majority of AQPs followed an increasing expression level trend with age. Of the three receptors/channels regulating ion transport in the murine cholangiocyte, only the cystic fibrosis transmembrane conductance regulator was found to follow a consistent trend of decreasing expression coincident with age. We have further validated AQP3 and AQP8 protein localization in both hepatocytes and cholangiocytes. This study emphasizes the need to further appreciate and consider the differences in cholangiocyte biology when treating neonatal and adult hepatobiliary diseases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Polycystic liver disease (PLD) is a member of the cholangiopathies, a group of liver diseases in which cholangiocytes, the epithelia lining of the biliary tree, are the target cells. PLDs are caused by mutations in genes involved in intracellular signaling pathways, cell cycle regulation, and ciliogenesis, among others. We previously showed that cystic cholangiocytes have abnormal cell cycle profiles and malfunctioning cilia. Because histone deacetylase 6 (HDAC6) plays an important role in both cell cycle regulation and ciliary disassembly, we examined the role of HDAC6 in hepatic cystogenesis. HDAC6 protein was increased sixfold in cystic liver tissue and in cultured cholangiocytes isolated from both PCK rats (an animal model of PLD) and humans with PLD. Furthermore, pharmacological inhibition of HDAC6 by Tubastatin-A, Tubacin, and ACY-1215 decreased proliferation of cystic cholangiocytes in a dose- and time-dependent manner, and inhibited cyst growth in three-dimensional cultures. Importantly, ACY-1215 administered to PCK rats diminished liver cyst development and fibrosis. In summary, we show that HDAC6 is overexpressed in cystic cholangiocytes both in vitro and in vivo, and its pharmacological inhibition reduces cholangiocyte proliferation and cyst growth. These data suggest that HDAC6 may represent a potential novel therapeutic target for cases of PLD., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Recent studies have demonstrated direct reprogramming of fibroblasts into a range of somatic cell types, but to date stem or progenitor cells have only been reprogrammed for the blood and neuronal lineages. We previously reported generation of induced hepatocyte-like (iHep) cells by transduction of Gata4, Hnf1α, and Foxa3 in p19 Arf null mouse embryonic fibroblasts (MEFs). Here, we show that Hnf1β and Foxa3, liver organogenesis transcription factors, are sufficient to reprogram MEFs into induced hepatic stem cells (iHepSCs). iHepSCs can be stably expanded in vitro and possess the potential of bidirectional differentiation into both hepatocytic and cholangiocytic lineages. In the injured liver of fumarylacetoacetate hydrolase (Fah)-deficient mice, repopulating iHepSCs become hepatocyte-like cells. They also engraft as cholangiocytes into bile ducts of mice with DDC-induced bile ductular injury. Lineage conversion into bipotential expandable iHepSCs provides a strategy to enable efficient derivation of both hepatocytes and cholangiocytes for use in disease modeling and tissue engineering., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background and Aim: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes., Methods: Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively., Results: Upon histomorphological analysis of cryo- and paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size., Conclusions: Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE.

Cholangiocytes are involved in a variety of processes essential for liver pathophysiology. To meet their demanding metabolic and functional needs, bile ducts are nourished by their own arterial supply, the peribiliary plexus. This capillary network originates from the hepatic artery and is strictly arranged around the intrahepatic bile ducts. Biliary and vascular structures are linked by a close anatomic and functional association necessary for liver development, normal organ physiology, and liver repair. This strong association is finely regulated by a range of angiogenic signals, enabling the cross talk between cholangiocytes and the different vascular cell types. This review will briefly illustrate the "vascular" properties of cholangiocytes, their underlying molecular mechanisms and the relevant pathophysiological settings., (© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Unlabelled: Large, but not small, cholangiocytes (1) secrete bicarbonate by interaction with secretin receptors (SRs) through activation of cystic fibrosis transmembrane regulator (CFTR), Cl(-) /HCO3 (-) (apex) anion exchanger 2 (Cl(-) /HCO3 (-) AE2), and adenylyl cyclase (AC)8 (proteins regulating large biliary functions) and (2) proliferate in response to bile duct ligation (BDL) by activation of cyclic adenosine monophosphate (cAMP) signaling. Small, mitotically dormant cholangiocytes are activated during damage of large cholangiocytes by activation of D-myo-inositol 1,4,5-trisphosphate/Ca(2+) /calmodulin-dependent protein kinase (CaMK) I. gamma-Aminobutyric acid (GABA) affects cell functions by modulation of Ca(2+) -dependent signaling and AC. We hypothesized that GABA induces the differentiation of small into large cholangiocytes by the activation of Ca(2+) /CaMK I-dependent AC8. In vivo, BDL mice were treated with GABA in the absence or presence of 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA/AM) or N-(6-aminohexyl)-5-chloro-1-naphtalenesulfonamide (W7) before evaluating apoptosis and intrahepatic bile ductal mass (IBDM) of small and large cholangiocytes. In vitro, control- or CaMK I-silenced small cholangiocytes were treated with GABA for 3 days before evaluating apoptosis, proliferation, ultrastructural features, and the expression of CFTR, Cl(-) /HCO3 (-) AE2, AC8, and secretin-stimulated cAMP levels. In vivo administration of GABA induces the apoptosis of large, but not small, cholangiocytes and decreases large IBDM, but increased de novo small IBDM. GABA stimulation of small IBDM was blocked by BAPTA/AM and W7. Subsequent to GABA in vitro treatment, small cholangiocytes de novo proliferate and acquire ultrastructural and functional phenotypes of large cholangiocytes and respond to secretin. GABA-induced changes were prevented by BAPTA/AM, W7, and stable knockdown of the CaMK I gene., Conclusion: GABA damages large, but not small, cholangiocytes that differentiate into large cholangiocytes. The differentiation of small into large cholangiocytes may be important in the replenishment of the biliary epithelium during damage of large, senescent cholangiocytes., (Copyright © 2013 American Association for the Study of Liver Diseases.)

We have previously demonstrated that disruption of polyubiquitin gene Ubc leads to mid-gestation embryonic lethality most likely due to a defect in fetal liver development, which can be partially rescued by ectopic expression of Ub. In a previous study, we assessed the cause of embryonic lethality with respect to the fetal liver hematopoietic system. We confirmed that Ubc(-/-) embryonic lethality could not be attributed to impaired function of hematopoietic stem cells, which raises the question of whether or not FLECs such as hepatocytes and bile duct cells, the most abundant cell types in the liver, are affected by disruption of Ubc and contribute to embryonic lethality. To answer this, we isolated FLCs from E13.5 embryos and cultured them in vitro. We found that proliferation capacity of Ubc(-/-) cells was significantly reduced compared to that of control cells, especially during the early culture period, however we did not observe the increased number of apoptotic cells. Furthermore, levels of Ub conjugate, but not free Ub, decreased upon disruption of Ubc expression in FLCs, and this could not be compensated for by upregulation of other poly- or mono-ubiquitin genes. Intriguingly, the highest Ubc expression levels throughout the entire culture period were observed in bipotent FLEPCs. Hepatocytes and bipotent FLEPCs were most affected by disruption of Ubc, resulting in defective proliferation as well as reduced cell numbers in vitro. These results suggest that defective proliferation of these cell types may contribute to severe reduction of fetal liver size and potentially mid-gestation lethality of Ubc(-/-) embryos., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Unlabelled: The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a β-catenin-independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1β(+) HNF4α(-) biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to WT liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady-state levels of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls., Conclusion: Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;)., (Copyright © 2013 American Association for the Study of Liver Diseases.)

TGR5, the G protein-coupled bile acid receptor that transmits bile acid signaling into a cell functional response via the intracellular cAMP signaling pathway, is expressed in human and rodent cholangiocytes. However, detailed information on the localization and function of cholangiocyte TGR5 is limited. We demonstrated that in human (H69 cells) and rat cholangiocytes, TGR5 is localized to multiple, diverse subcellular compartments, with its strongest expression on the apical plasma, ciliary, and nuclear membranes. To evaluate the relationship between ciliary TGR5 and the cholangiocyte functional response to bile acid signaling, we used a model of ciliated and nonciliated H69 cells and demonstrated that TGR5 agonists induce opposite changes in cAMP and ERK levels in cells with and without primary cilia. The cAMP level was increased in nonciliated cholangiocytes but decreased in ciliated cells. In contrast, ERK signaling was induced in ciliated cholangiocytes but suppressed in cells without cilia. TGR5 agonists inhibited proliferation of ciliated cholangiocytes but activated proliferation of nonciliated cells. The observed differential effects of TGR5 agonists were associated with the coupling of TGR5 to Gαi protein in ciliated cells and Gαs protein in nonciliated cholangiocytes. The functional responses of nonciliated and ciliated cholangiocytes to TGR5-mediated bile acid signaling may have important pathophysiological significance in cilia-related liver disorders (i.e., cholangiociliopathies), such as polycystic liver disease. In summary, TGR5 is expressed on diverse cholangiocyte compartments, including a primary cilium, and its ciliary localization determines the cholangiocyte functional response to bile acid signaling.

End-stage liver diseases represent major health problems that are currently treated by liver transplantation. However, given the world-wide shortage of donor livers novel strategies are needed for therapeutic treatment. Adult stem cells have the ability to self-renew and differentiate into the more specialized cell types of a given organ and are found in tissues throughout the body. These cells, whose progeny are termed progenitor cells in human liver and oval cells in rodents, have the potential to treat patients through the generation of hepatic parenchymal cells, even from the patient's own tissue. Little is known regarding the nature of the hepatic progenitor cells. Though they are suggested to reside in the most distal part of the biliary tree, the canal of Hering, the lack of unique surface markers for these cells has hindered their isolation and characterization. Upon activation, they proliferate and form ductular structures, termed "ductular reactions", which radiate into the hepatic parenchyma. The ductular reactions contain activated progenitor cells that not only acquire a phenotype resembling that observed in developing liver but also display markers of differentiation shared with the cholangiocytic or hepatocytic lineages, the two parenchymal hepatic cell types. Interactions between the putative progenitor cells, the surrounding support cells and the extracellular matrix scaffold, all constituting the progenitor cell niche, are likely to be important for regulating progenitor cell activity and differentiation. Therefore, identifying novel progenitor cell markers and deciphering their microenvironment could facilitate clinical use. The aims of the present PhD thesis were to expand knowledge of the hepatic progenitor cell niche and characterize it both during development and in disease. Several animal models of hepatic injury are known to induce activation of the progenitor cells. In order to identify possible progenitor cell markers and niche components, we examined several genes upregulated in a global gene expression array conducted on one of these models, in which progenitor cells are activated. The protein expression patterns were evaluated in our collections of human embryonic and fetal livers, human liver diseases, and rodent hepatic injury models. When analyzing standard histological liver sections underlying connections and tissue architecture are not immediately evident. We therefore developed models for digitally reconstructing not only protein expression in serially cut tissue sections, but also vessels of the portal area. Article I constituted our earliest attempts to create 3D reconstructions of biological material. Human embryonic stem cell cultures were previously thought to consist of homogenously undifferentiated cells. The protocols for 3D reconstructions developed in this study demonstrated micro heterogeneity in expression of differentiation markers and provided the basis for later reconstructions of hepatic tissues. In article II we examined the expression patterns of chosen proteins seen upregulated in the gene array as well as classical hepatocytic and cholangiocytic markers in human liver disease and during prenatal development. Previous studies had indicated direct connections between activated progenitor cells apparently isolated in the parenchyma and the intrahepatic biliary tree. Our developed protocols for 3D reconstructions visually demonstrated direct connections between these entities. Analysis of protein expression in prenatal liver revealed the formation of the intrahepatic tree to occur through a special form of asymmetric tubulogenesis, only recently described in mice. In order to describe the composition of the hepatic progenitor cell niche and the localization of cell surface proteins in article III, the expression patterns of certain genes upregulated in the gene array analysis were analyzed in different models of rodent liver regeneration. We observed that the extracellular matrix molecules collagen 1a1, laminin, nidogen-1 and agrin embraced the biliary cells and sharply defined the hepatic progenitor cell niche, which was encircled by desmin positive support cells. In all injury models biliary cells expressed the cell surface proteins matriptase and HAI-1. However, in the so-called 2-AAF/PHx model of progenitor cell activation, a subpopulation of hepatic progenitor cells was positive for Dlk1. 3D reconstructions clarified that the Dlk1-subpopulation was entirely located in the portal area periphery, and connected to the bile ducts via HAI-1 positive biliary cells. The heterogeneous expression patterns of matriptase, HAI-1 and Dlk1 in this particular injury model indicate the presence of a cellular hierarchy containing possibly less differentiated Dlk1-positive hepatic progenitor cells. In conclusion, our studies characterized the hepatic progenitor cell niche in humans and rodents. We successfully developed protocols for digitally visualizing, not only hepatic, but virtually any tissue through two fundamentally distinct approaches. The identification of an asymmetric form of tubulogenesis in humans added new knowledge to the development of the intrahepatic biliary tree, and thereby the formation of the progenitor cell niche. The identification of heterogeneously expressed cell surface proteins and extracellular matrix components provided knowledge of the constituents defining the niche. These pieces of information are important for future isolation and characterization studies of biliary subpopulations and their differentiation abilities in vitro.

Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine.

The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the 'eat-me' signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis., (© 2012 British Society for Immunology.)