Regional Differences in Human Biliary Tissues and Corresponding In Vitro-Derived Organoids.

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.

Oncogenic KRAS-expressing organoids with biliary epithelial stem cell properties give rise to biliary tract cancer in mice.

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.

Unique Cholangiocyte-Targeted IgM Autoantibodies Correlate With Poor Outcome in Biliary Atresia.

BACKGROUND AND AIMS: The etiology of biliary atresia (BA) is not known and is likely multifactorial, including a genetic predisposition, a viral or environmental trigger, an aberrant autoimmune response targeting cholangiocytes, and unique susceptibilities of the neonatal bile ducts to injury. Damaged cholangiocytes may express neo self-antigens and elicit autoreactive T-cell-mediated inflammation and B-cell production of autoantibodies. The aim of this study was to discover autoantibodies in BA that correlated with outcomes. APPROACH AND RESULTS: An autoantigen microarray encompassing approximately 9,500 autoantigens was used to screen for serum immunoglobulin M (IgM) and immunoglobulin G (IgG) autoantibodies in patients with BA or other liver disease controls. Validation of candidate autoantibodies by enzyme-linked immunosorbent assay on a second cohort of subjects (6-12 months following Kasai portoenterostomy) and correlations of autoantibodies with outcomes were performed (serum bilirubin levels and need for liver transplant in first 2 years of life). Mean anti-chitinase 3-like 1 (CHI3L1), anti-delta-like ligand (DLL-4), and antisurfactant protein D (SFTPD) IgM autoantibodies in BA were significantly higher compared with controls, and IgM autoantibody levels positively correlated with worse outcomes. Immunofluorescence revealed cholangiocyte-predominant expression of CHI3L1, DLL-4, and SFTPD. The humoral autoantibody response was associated with C3d complement activation and T-cell autoimmunity, based on detection of cholangiocyte-predominant C3d co-staining and peripheral blood autoreactive T cells specific to CHI3L1, DLL-4 and SFTPD, respectively. CONCLUSIONS: BA is associated with cholangiocyte-predominant IgM autoantibodies in the first year after Kasai portoenterostomy. Anti-CHI3L1, anti-DLL-4, and anti-SFTPD IgM autoantibody correlations with worse outcomes and the detection of C3d on cholangioctyes and antigen-specific autoreactive T cells suggest that autoimmunity plays a role in the ongoing bile duct injury and progression of disease.

Coordinated development of the mouse extrahepatic bile duct: Implications for neonatal susceptibility to biliary injury.

BACKGROUND & AIMS: The extrahepatic bile duct is the primary tissue initially affected by biliary atresia. Biliary atresia is a cholangiopathy which exclusively affects neonates. Current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts. METHODS: We studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes, as well as isolated extrahepatic bile ducts, and a collagen reporter mouse. The methods used included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix. RESULTS: The cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling of the matrix suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen. CONCLUSION: We identified 4 key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis. LAY SUMMARY: Biliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction, resulting in liver injury. We identify 4 key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.

Generation of Organoids from Mouse Extrahepatic Bile Ducts.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

Single cell RNA sequencing reveals regional heterogeneity of hepatobiliary innate lymphoid cells in a tissue-enriched fashion.

IL-33 promotes type 2 immunity, epithelial repair, and tissue fibrosis by activating group 2 innate lymphoid cells (ILC2). ILC2 lack all known surface markers of mature T, B, NK, and myeloid cell lineages (Linneg), express the IL-33 receptor ST2, and release type 2 cytokines which contribute to cholangiocyte proliferation and activation of hepatic stellate cells. This pathway results in massive proliferation of the extrahepatic bile duct (EHBD) but also exacerbates liver fibrosis, suggesting that there may be tissue-specific subpopulations of IL-33-induced ILC. To determine the tissue-specific subsets of ILC in the hepatobiliary system, we analyzed CD45+Linneg mononuclear cells from IL-33 treated adult Balb/c mouse liver or EHBD by single cell RNA sequencing. Principal component analysis identified 6 major CD45+Linneg cell classes, two of which were restricted to the EHBD. One of these classes, biliary immature myeloid (BIM) cells, was predicted to interact with ILC2 by a network of shared receptor-ligand pairs. BIM highly expressed Gp49 and ST2 receptors on the cell surface while lacking surface expression of markers for mature myeloid cells. In conclusion, single cell RNA sequencing identified IL-33 responsive cell groups regionally confined to the liver or extrahepatic bile duct, including a novel population of CD45+Linneg Gp49-expressing mononuclear cells.

Characterization of Peribiliary Gland-Constituting Cells Based on Differential Expression of Trophoblast Cell Surface Protein 2 in Biliary Tract.

Peribiliary glands (PBGs) are accessory glands with mucinous and serous acini in the biliary tree. The PBG is composed of a heterogeneous cell population, such as mucus- and pancreatic enzyme-producing epithelial cells, whereas it constitutes niches for multipotential stem/progenitor cells in the human extrahepatic bile duct (EHBD). By contrast, the nature of PBGs in the mouse EHBD remains unclear. Our aim was to establish a method for isolating and characterizing PBG-constituting cells in the mouse EHBD. We found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. On the basis of the differential expression profile of Trop2, lumen-forming biliary epithelial cells (LBECs) and PBG-constituting epithelial cells (PBECs) were separately isolated for further characterization. Gene expression analysis revealed that the isolated mouse PBECs expressed several marker genes related to human PBGs. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. In the organoid formation assay, PBECs formed cystic organoid with LBEC-like phenotype. Interestingly, PBECs proliferated, accompanied by reexpression of Trop2 in vivo after bile duct ligation. Furthermore, the unique expression profile of Trop2 was conserved in human EHBD. Our findings indicate that Trop2 is a useful marker in investigating the pathophysiological roles and characteristics of mouse and human PBGs in biliary diseases.

Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

Development and functional characterization of extrahepatic cholangiocyte lines from normal rats.

BACKGROUND: Since limited in vitro tools exist for evaluating the pathophysiology of extrahepatic bile ducts, we aim to develop an extrahepatic cholangiocyte culture system from normal rats. METHODS: Extrahepatic ducts were dissected from rats, cut in half length-wise and cultured on collagen-I coated plates. Transepithelial electrical resistance was measured. At ∼85% confluence, in extrahepatic cholangiocytes we measured: (i) cell size and distribution, and expression for cytokeratin-19, secretin, secretin receptor and somatostatin receptor type II (SSTR2), cystic fibrosis transmembrane conductance regulator (CFTR), chloride bicarbonate anion exchanger 2 (AE2), vascular endothelial growth factor-A (VEGF-A) and nerve growth factor (NGF); and (ii) the effect of secretin and/or somatostatin on 3'-5'-cyclic adenosine monophosphate (cAMP) levels and proliferation. RESULTS: Cytokeratin-positive extrahepatic cholangiocytes were cultured for 6 passages to form a cell monolayer. Cholangiocytes proliferated to confluence over a 2-week period. The size of extrahepatic cholangiocytes averaged ∼16 µm. Extrahepatic ducts and cholangiocytes were positive for secretin, secretin receptor and SSTR2, CFTR, AE2, VEGF-A and NGF. In extrahepatic cholangiocyte cultures, secretin increased cAMP (prevented by somatostatin), chloride efflux and proliferation. CONCLUSIONS: Extrahepatic cholangiocyte cultures may be important for studying diseases targeting extrahepatic cholangiocytes such as biliary atresia.

Isolation of neonatal extrahepatic cholangiocytes.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood. Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established(1-4). Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts. This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

Multipotent stem/progenitor cells in the human foetal biliary tree.

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

Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.

BACKGROUND & AIMS: Although regeneration of intrahepatic bile ducts has been extensively studied and intrahepatic progenitor cells have been identified, few studies have focussed on the extrahepatic bile duct (EHBD). We hypothesized that local progenitor cells are present within the EHBD of humans. Human EHBD specimens (n = 17) were included in this study. METHODS: Specimens of normal EHBD tissue were obtained from healthy donor livers (n = 6), mildly injured EHBD from patients with cholangitis (n = 6) and severely injured EHBD from patients with ischaemic type biliary lesions (n = 5). Double immunostaining for K19 and the proliferation marker Ki-67 was performed to identify and localize proliferating cells. In addition, immunofluorescent doublestaining using antibodies against K19 and c-Kit was performed to identify and localize cholangiocytes co-expressing putative progenitor cell markers. RESULTS: In normal EHBD, few Ki-67(+) cells were detected, whereas large numbers of Ki-67(+) were found in the diseased EHBD. In EHBD affected by cholangitis, Ki-67(+) cells were mainly located in the basal layer of the lumen. EHBD specimens from patients with ischaemic type biliary lesions displayed histological signs of epithelial cell loss and large numbers of Ki-67(+) cells were observed in the peribiliary glands. C-Kit expression was localized throughout the EHBD wall and immunofluorescent doublestaining identified a few K19(+) /c-Kit(+) cells in the luminal epithelium of the EHBD as well as in the peribiliary glands. CONCLUSIONS: These findings support the hypothesis that progenitor cells exist in the EHBD and that the peribiliary glands can be considered a local progenitor cell niche in the human EHBD.

Biliary tree stem/progenitor cells in glands of extrahepatic and intraheptic bile ducts: an anatomical in situ study yielding evidence of maturational lineages.

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo-alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co-expressing liver and pancreatic markers (e.g. PDX1(+)/SOX17(+)) are EpCAM(+/-), and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis.

Multipotent stem cells in the biliary tree.

The biliary tree system consists of two divisions: intrahepatic bile ducts and extrahepatic bile ducts. The development of the biliary tree, and secondarily the liver, shares a common origin with ventral pancreas. A common progenitor for liver, biliary duct system, and ventral pancreas exists at early stages of development, when the anterior definitive endoderm is forming the foregut. Several studies indicate that the biliary tree contains stem cell compartments for liver, pancreas and the bile duct system and persisting into adulthood. These stem cell compartments are present in the peribiliary glands and possibly give rise to committed progenitors in gallbladder that does not have peribiliary glands. The biliary tree stem/progenitors represent a new source of cells that can be used as tools for regenerative medicine of liver, bile duct and pancreas.

A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice.

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts' epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO(2). The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5-6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrane, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.

Interstitial cells of Cajal are present in human extrahepatic bile ducts.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are distributed with smooth muscle throughout the gastrointestinal tract and are involved in regulating motility. ICC were recently discovered in the wall of the human gallbladder. This study sought to determine whether ICC are present in human bile ducts. METHODS: Biliary tract samples were obtained from several sources: surgical specimens (n = 16, 11 women, mean age 61 years); archival post-mortem specimen (n = 1, 86 years, man); and cadavers (n = 2, 68 and 80 years, men). Paraffin-embedded sections (3 microm) from the gallbladder (fundus, body and neck) and both extrahepatic and intrahepatic bile ducts were investigated. A double immunofluorescence protocol using polyclonal and monoclonal c-kit antibodies and mast cell tryptase was used to distinguish c-kit-positive cells with typical ICC morphology from c-kit-positive mast cells. Small bowel samples were used as positive controls. ICC in the gallbladder were confirmed by ultrastructural study. RESULTS: c-kit-positive cells with characteristic ICC morphology were identified in the subepithelial and muscular layers of the gallbladder and extrahepatic bile ducts. They were most prominent within the muscle layer of the extrahepatic bile ducts where they were organized into loosely arranged laminae running parallel to circular smooth muscle fibers. ICC were not found in intrahepatic bile ducts. CONCLUSION: This study demonstrates for the first time that ICC are present in human extrahepatic bile ducts where they are more densely aggregated than in the gallbladder. This cellular network is likely to be involved in biliary tract motility and its related disorders.

Cholangiocyte secretion of chemokines in experimental biliary atresia.

UNLABELLED: Biliary atresia (BA) is a disease of the newborn that results in obstruction of the biliary tree. The cause of BA remains unknown; however, recent studies using the murine model of biliary atresia have found that rotavirus infection of the biliary epithelial cell (cholangiocyte) triggers an inflammatory response. We hypothesized that rotavirus infection of cholangiocytes results in the release of chemokines, important mediators of the host immune response. METHODS: In vivo, Balb/c pups were injected with rhesus rotavirus (RRV) or saline, and, their extrahepatic bile ducts were microdissected 2, 5, 7, and 14 days after injection. Next, an immortalized cholangiocyte cell line (mCl) was incubated with RRV or serum-free media. Qualitative and quantitative chemokine assessment was performed using enzyme-linked immunosorbent assay, polymerase chain reaction, and immunohistochemistry. RESULTS: In vivo, increased levels of the chemokines macrophage inflammatory protein 2, monocyte chemotactic protein 1, KC and Regulated upon Activation, Normal T Expressed and Secreted were found in RRV-infected murine bile ducts. In vitro, infected mCl cells produced increasing amounts of these same chemokines in relation to dose and time. CONCLUSION: These novel results suggest that chemokine expression by RRV-infected cholangiocytes may trigger a host inflammatory process that causes bile duct obstruction. Understanding how viral infection initiates this response may shed light on the pathogenesis of biliary atresia.

Tbx3 promotes liver bud expansion during mouse development by suppression of cholangiocyte differentiation.

UNLABELLED: After specification of the hepatic endoderm, mammalian liver organogenesis progresses through a series of morphological stages that culminate in the migration of hepatocytes into the underlying mesenchyme to populate the hepatic lobes. Here, we show that in the mouse the transcriptional repressor Tbx3, a member of the T-box protein family, is required for the transition from a hepatic diverticulum with a pseudo-stratified epithelium to a cell-emergent liver bud. In Tbx3-deficient embryos, proliferation in the hepatic epithelium is severely reduced, hepatoblasts fail to delaminate, and cholangiocyte rather than hepatocyte differentiation occurs. Molecular analyses suggest that the primary function of Tbx3 is to maintain expression of hepatocyte transcription factors, including hepatic nuclear factor 4a (Hnf4a) and CCAAT/enhancer binding protein (C/EBP), alpha (Cebpa), and to repress expression of cholangiocyte transcription factors such as Onecut1 (Hnf6) and Hnf1b. CONCLUSION: Tbx3 controls liver bud expansion by suppressing cholangiocyte and favoring hepatocyte differentiation in the liver bud.

Distribution of the interstitial Cajal-like cells in the gallbladder and extrahepatic biliary duct of the guinea-pig.

It has been suggested that interstitial Cajal-like cells (ICLC) may be involved in the spontaneous rhythmic electrical activities of the extrahepatic bile duct system. The present study investigated the distribution and characteristics of ICLC, which are immunopositive for CD117/ Kit receptor tyrosine kinase, using immunohistochemistry employing a monoclonal antibody raised against CD117/Kit on whole-mount preparations. The Kit-positive ICLC were examined using confocal laser scanning microscopy or fluorescence microscopy. ICLC, immunoreactive for Kit, were pleiomorphic and/or spindle-shaped cells with a few bipolar processes and distributed in the smooth muscle layers of the gallbladder and bile duct system. They were scattered in the hepatic duct, cystic duct and gallbladder as well as in the upper part of the common bile duct. The ICLC gradually increased in number and formed a completed cellular network in the lower part of the common bile duct and ampulla. The numbers of ICLC in the ampulla were similar to that of the duodenum and significantly much greater in number than in the gallbladder and bile ducts. The density of the ICLC in the common bile duct was significantly higher than that of other bile ducts. Our results suggested that the ICLC might contribute to the regulation of the spontaneous rhythmic contraction and development of motility disorders of the bile duct system.