Regional differences in human biliary tissues and corresponding in vitro derived organoids
ABSTRACT: RNA-Sequencing was performed on mechanically dissociated, epithelial-enriched samples, of human extrahepatic biliary tissue from Gallbladder, Common Bile Duct, and Pancreatic Duct tissues. Sequencing was also performed on in vitro cultures of Organoid cell lines at passage 5 that were derived from human Gallbladder, Common Bile Duct, Pancreatic Duct, or Intrahepatic Bile Ducts.
Project description:Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine and colon. Here we derived organoids from mouse gallbladder tissue (gallbladder organoids), from mouse liver (including the extrahepatic biliary ducts and gallbladder; liver organoids) and from mouse small intestine tissue (intestinal organoids). RNA was prepared from these organoids and used to assay expression of 21,258 genes using Affymetrix gene expression arrays. RNA was also prepared from mouse gallbladder, liver and small intestine tissues and used to assay gene expression in these tissues. Finally, gallbladder organoids were induced to differentiate by removing R-spondin 1 and noggin from the culture media and subjected to gene expression array analysis. RNA was extracted from mouse gallbladder organoids, differentiated gallbladder organoids, liver organoids, small intestine organoids, gallbladder tissue, liver tissue and small intestine tissue and then used for hybridization of Affymetrix gene expression microarrays.
Project description:Sox17 expression is important for development of gallbladder and bile duct systems in embryo, and it is reported that gallbladder hypoplasia in Sox17 hetero genic embryo. Additionally it was reported that hepatitis was occurred in Sox17 hetero genic newborn by gallbladder hypoplasia. So, we examined Sox17 gene cascade and the role for the formation of gallbladder and bile duct systems by microarray analysis on Sox17 hetero genic gallbladder in day 15 of pregnancy when Sox17 express and gallbladder epithelium alternated morphology. We detected that expression of Sonic hedgehog (shh) signal genes decreased in sox17+/- gallbladder and cysticduct as compared with the wildtype gallbladders.These arry analysis in gallbladder and cysticduct reveal expression of shh in developmental gallbladder is downstream in sox17+/- and gene expression in sox17+/- gallbladder was similar in cysticduct . Overall design: Total RNAs from gallbladder and cysticduct of wildtype and Sox17+/- mice at 15dpc were subjected to microarray analyses. Two biological replicates of each genotype were analyzed.
Project description:Despite the impact of bile duct disorders, treatment options remain very limited. Poor access to biliary tissue and restrictions in long-term culture or significant expansion of primary cholangiocytes have posed major challenges for research in the field. These limitations have so far precluded large scale experiments such as transcriptomic and genome-wide analyses which are urgently needed to better understand biliary physiology and pathophysiology. To address this issue, we have developed a novel system for the isolation and propagation of primary cholangiocytes from the extrahepatic bile ducts. The resulting Extrahepatic Cholangiocyte Organoids (ECOs) maintain their genetic stability, transcriptomic profile and function over long term culture and are compatible with regenerative medicine applications such as biliary reconstruction. We established a novel protocol for the isolation and propagation of primary cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs). The aim of this experiment was to provide in depth characterisation of the transcriptome of ECOs during long term culture. We compare the transcriptome of ECOs cultured for 1 passage (P1), 10 passages (P10) and 20 passages (P20) with freshly isolated primary cholangiocytes from the common bile duct. Embryonic Stem Cells (ES) cells are used as a negative control=
Project description:Infection with Salmonella enterica serovar Typhi in humans causes the systemic, life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. The ensuing disease is characterized by systemic dissemination and colonization of many organs, including the liver, spleen and gallbladder. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment we compared the transcriptome of Salmonella cultures grown in LB or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of phoP as a bile-responsive gene. Repression of phoP expression does not involve PhoPQ sensing of a bile component. Due to its critical role in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease. Salmonella enterica serovar Typhimurium LT2 glass slide microarrays were obtained through the NIAID’s Pathogen Functional Genomics Resource Center, managed and funded by Division of Microbiology and Infectious Diseases, NIAID, NIH, DHHS and operated by the J. Craig Venter Institute. Two independent cultures of Salmonella enterica serovar Typhimurium SL1344 were grown overnight in Luria-Bertani (LB) broth with streptomycin (100 μg/mL) at 37oC with shaking (225 RPM). Cells were pelleted by centrifugation and resuspended in sterile Dulbecco’s phosphate-buffered saline (PBS). These suspensions were further diluted 1:50 in PBS and used to inoculate 70 μL of either LB or physiological murine bile (in 0.65-mL tubes) at a 1:100 dilution. Cultures were incubated for 24 hours at 37oC with agitation (225 RPM). Then, samples were diluted in PBS and serial dilutions were plated on LB plates containing 100 μg/mL of streptomycin and incubated overnight at 37oC for bacterial growth and enumeration by colony counting. The remaining cells were used in the subsequent steps, as described below. RNA isolation began with the addition of 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to the bacterial cultures and incubation at room temperature for approximately 5 minutes. Cells were pelleted by centrifugation and RNA was isolated using the RNeasy Mini Kit (Qiagen) with the on-column DNase digestion, according to the manufacturer’s recommendations. Synthesis of cDNA was performed as described herein. The entire RNA sample (≥3 μg) was mixed with 6 μg of Random Primers (Invitrogen, Burlington, Canada) and 40 units of RNaseOUT recombinant ribonuclease inhibitor (Invitrogen, Burlington, Canada) in a total volume of 18.5 μL and incubated at 70oC for 10 minutes. Samples were incubated on ice for 30 seconds, centrifuged to bring down condensation and incubated with the following components (final concentrations; Invitrogen): First Strand Buffer (1X), dithiothreitol (DTT; 10 mM), dNTPs (dATP, dCTP, dGTP, dTTP; 0.5 mM each) and 100 units of SuperScript II reverse transcriptase in a total volume of 30 μL. The mixture was incubated at 42oC overnight and the reaction was stopped and the RNA degraded through the addition of 0.5 M EDTA and 1 M sodium hydroxide (10 μL each) and incubation at 65oC for 15 minutes. Then, 25 μL of 1 M Tris (pH 7) was added to neutralize the pH of the cDNA solution. cDNA was purified using the MinElute PCR Purification Kit (Qiagen) according to the manufacturer’s recommendations, except that the Qiagen wash and elution buffers were substituted by phosphate buffers, according to the PFGRC microarray protocol. cDNA was then precipitated using ammonium acetate and ethanol and labeled with Cy3 (LB cultures) and Cy5 (bile cultures) using the ULS aRNA Fluorescent Labeling Kit (KREATECH Biotechnology, Amsterdam, The Netherlands) according to the manufacturer’s instructions. Samples were mixed, dried and saved at -80oC until used. Pre-hybridization, hybridization and washing steps were performed essentially as described in the PFGRC protocols, except that the hybridization buffer contained KREAblock blocking buffer (25%; KREATECH Biotechnology). After hybridization, slides were scanned using an Affymetrix 428 Array Scanner from Eurofins MWG Operon (Huntsville, USA).
Project description:Liver dysfunction and cirrhosis affect vasculature in several organ systems and cause impairment of organ functions, thereby increasing morbidity and mortality. If a mouse model of hepatopulmonary syndrome (HPS) could be established, greater insight into the genetic basis of the disease would be gained. Our objectives were to establish a mouse model of lung injury after common bile duct ligation (CBDL) and to investigate pulmonary pathogenesis for application in future therapeutic approaches. Balb/c mice were subjected to CBDL. Immunohistochemical analyses and real-time quantitative reverse transcriptional polymerase chain reaction were performed on pulmonary tissues. The presence of HPS markers were detected by western blot and microarray analyses. We observed extensive proliferation of CD31-positive pulmonary vascular endothelial cells 2 weeks after CBDL, and identified 11 up-regulated and 8 down-regulated proteins that were associated with angiogenesis. MMP-9 protein was highly expressed at 3 weeks after CBDL, and less expressed in lungs of the control group. Contrary to our expectation, lung pathology in our mouse model exhibited differences from that of rat models, and the mechanisms responsible for these differences are unknown. This phenomenon may be explained by contrasting processes related to TNF induction of angiogenic signaling pathways in the inflammatory phase; thus, we suggest that our mouse model can be applied to pulmonary pathological analyses in the inflammatory phase, i.e., to systemic inflammatory response syndrome, acute lung injury, and MOD syndrome. After induction of anesthesia, a median abdominal incision was made and the common bile duct was identified. The duct was dissected carefully under a microscope, and doubly ligated with 7-0 Prolene and transected. In the sham operation (control) group, the duct was dissected without common bile duct ligation. Mice were sacrificed at 2 and 3 weeks after surgery. CD31-positive cells were assembled from three mice in each group.
Project description:Decreased bile secretion in rodents by either ligation of the common bile duct or induction of cirrhosis causes changes in the small intestine, including bacterial overgrowth and translocation across the mucosal barrier. Oral administration of bile acids inhibits these effects. The genes regulated by FXR in ileum suggested that it might contribute to the enteroprotective actions of bile acids. To test this hypothesis, mice were administered either GW4064 or vehicle for 2 days and then subjected to bile duct ligation (BDL) or sham operation. After 5 days, during which GW4064 or vehicle treatment was continued, the mice were killed and their intestines were analyzed for FXR target gene expression. Mice were treated with or without FXR ligand GW4064 for 2 days prior to bile duct ligation surgery and for 5 days after surgery. After 5 days the mice were sacrificed and the ileum collected and processed for gene expression analysis. Gene expression in the ilium from each sample group was assayed in duplicate using Affymetrix Mouse Genome 430A 2.0 Gene Chips.
Project description:Genome wide DNA methylation profiling of normal and tumor bile duct samples. The Illumina HumanMethylation450 BeadChip was used to obtain DNA methylation profiles across approximately 450,000 CpGs in 138 tumor bile duct samples and 4 normal bile duct samples. Overall design: Bisulphite converted DNA from the 142 samples were hybridised to the Illumina HumanMethylation450 BeadChip
Project description:The HGF/c-Met system is an essential inducer of hepatocyte growth and proliferation. Although a fundamental role for the HGF receptor c-Met has been demonstrated in acute liver regeneration its cell specific role in hepatocytes during chronic liver injury and fibrosis progression has not been determined yet. In order to better characterize the role of c-Met in hepatocytes we generated a hepatocyte-specific c-Met knockout mouse (c-Met∆hepa) using the Cre-loxP system and studied its relevance after bile-duct ligation. Two strategies for c-Met deletion in hepatocytes were tested. Early deletion during embryonic development was lethal, while post-natal Cre-expression was successful leading to the generation of viable c-Met∆hepa mice. Bile-duct ligation in these mice resulted in extensive necrosis and lower proliferation rates of hepatocytes. Gene array analysis of c-Met∆hepa mice revealed a significant reduction of anti-apoptotic genes in c-Met deleted hepatocytes. These findings could be functionally tested as c-Met∆hepa mice showed a stronger apoptotic response after bile-duct ligation and Jo-2 stimulation. This phenotype was associated with increased expression of pro-inflammatory cytokines (TNF-a and IL-6) and an enhanced recruitment of neutrophils. Activation of these mechanisms triggered a stronger pro-fibrogenic response as evidenced by increased TGF-b1, a-SMA, collagen-1a mRNA expression and enhanced collagen-fiber staining in c-Met∆hepa mice. For gene array analysis c-MetDhepa and c-MetloxP/loxP controls were stimulated for 2 hours with 2µg recombinant mouse HGF.Three animals per group were treated in parallel, before and after i.p. injection of recombinant HGF or NaCl.
Project description:To identify miRNAs differentially expressed in cholangiocarcinoma,3 human cholangiocarcinoma and their corresponding normal bile duct tissues were obtained from 3 patients after operation with postoperative pathological diagnosed perihilar or distal biliary cholangiocarcinoma miRNAs expression in human cholangiocarcinoma/normal bile duct samples was measured after operation.Three independent experiments were performed using different patients for each experiment.
Project description:Assays in bile duct cancer patients showed 984 CNVs in 306 CNV regions (CNVR) distributed throughout all 22 chromosomes. Bile duct cancer patients had a mean of 21.8 gains and 19.2 losses of genes, with an average of 35.9 CNVRs per patient. Frequent sites of gains were at chromosomes 22q11.22, 2p11.2-p.11.1, 14q32.33 and 17q12, whereas frequent sites of losses were at 19q12-q13.43. Investigation of CNV in 24 bile duct cancer tissue samples