Bile Duct Epithelial Cells Research Articles

Effect of Clonorchis sinensis and N-nitrosodimethylamine on host cell proliferation and modulation of cancer related moleclues (P1356)

Abstract Clonorchis sinensis (C. sinensis) infection is known to cause cholangiocarcinomas in humans, but the mechanism is not clearly understood. In order to verify the role of excretory/secretory products (ESP) from C. sinensis in cell proliferation and the modulation of cancer related molecules, HEK293T cells were cultured with ESP and/or NDMA (N-nitrosodimethylamine), and then the mode of cell cycle and the expression of proteins were analyzed by using FACS, western blotting and confocal analysis. When the ESP of C. sinensis and NDMA were treated, the degree of cell proliferation was upregulated and the proportion of G2/M phase cells were increased by 25%-30% as compared with that of control. The expression pattern of the cell cycle proteins was also changed when both ESP and NDMA were added. Theses indicate that ESP from C. sinensis and NDMA synergistically affected the regulation of cell cycle proteins. In addition, the levels of Cox-2 and connexin 43 (Cx43) which is known as cancer related gap junction protein were upregulated by the addition of ESP and NDMA in vivo. In addition, the level of another gap junction protein, Cx32, which is also known as cancer inhibitor was down- regulated. Our results suggest that exposure to C. sinensis and a small amount of NDMA promote uncontrolled cellular proliferation of the bile duct epithelial cells and the upregulation of cancer related intracellular molecules.

Treatment of Metastatic Renal Cell Carcinoma With CAIX CAR-engineered T cells: Clinical Evaluation and Management of On-target Toxicity

Autologous T cells genetically modified to express a chimeric antibody receptor (CAR) against carboxy-anhydrase-IX (CAIX) were administered to 12 patients with CAIX-expressing metastatic renal cell carcinoma (RCC). Patients were treated in three cohorts with a maximum of 10 infusions of a total of 0.2 to 2.1 × 10(9) CAR T cells. CTC grade 2-4 liver enzyme disturbances occurred at the lowest CAR T cell doses, necessitating cessation of treatment in four out of eight patients in cohorts 1 and 2. Examination of liver biopsies revealed CAIX expression on bile duct epithelium with infiltration of T cells, including CAR T cells. Subsequently four patients were pre-treated with CAIX monoclonal antibody (mAb) G250 to prevent CAR-specific toxicity and showed no liver toxicities and indications for enhanced peripheral T cell persistence. No clinical responses were recorded. This report shows that CAIX-targeting CAR T cells exerted antigen-specific effects in vivo and induced liver toxicity at the lowest dose of 0.2 × 10(9) T cells applied, illustrating the potency of receptor-modified T cells. We provide in-patient proof that the observed "on-target" toxicity is antigen-directed and can be prevented by blocking antigenic sites in off-tumor organs and allowing higher T cell doses.

Induction of cholangiocarcinoma cells apoptosis by an andrographolide analog

Cholangiocarcinoma (CCA) is a malignant tumor derived from bile duct epithelial cells that has high incidence in the Northeastern area of Thailand. It is associated with the infection of liver fluke (Opisthorchis viverrini). Chemotherapeutic treatment of CCA is often ineffective due to its resistance to drugs. Therefore, new effective agents for treatment of CCA are urgently required. RSPP 050 analog is a modified compound of andrographolide which has previously been reported to potently inhibit proliferation in many cancer cell lines. In the present study, we aim to investigate the anti-proliferative effect of RSPP 050 on cholangiocarcinoma cells (KKU-M213). Cell viability of KKU-M213 was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC50 values were 9.4±3.12 µM, 3.47±0.75 µM, and 3.44±0.5 µM at 24, 48 and 72 h, respectively. To investigate the induction of apoptosis in KKU-M213 cells by RSPP 050, APO-BrdU™ TUNEL assay was conducted. RSPP 050 markedly increased the number of apoptotic cells compared to the untreated control. By using 4'-6-diamidino-2-phenylindole(DAPI) staining, RSPP 050 at 10 µM for 24 h, increased the chromatin condensation about 25±0.12% compared with 0.1% DMSO treated cells (2±0.04%) and the percentage of nuclear fragmentation of 50±0.33% compared to 0.1% DMSO treated cells (4±0.01%). These results suggest that RSPP 050 potently inhibited proliferation of KKU-M213 cells by inducing apoptosis. It would be useful for further development as the therapeutic agent for treatment of cholangiocarcinoma. Key words: Andrographolide analog, apoptosis, cholangiocarcinoma.

Differential effects of norUDCA and UDCA in obstructive cholestasis in mice

Background & AimsThe quest for effective drugs to treat cholangiopathies led to the development of norUDCA previously shown to have potent choleretic effects and to heal cholangiopathy in Abcb4 knockout (Abcb4−/−) mice. Its mother compound UDCA had detrimental effects in common bile duct ligated (CBDL) mice, presumably related to its choleretic effects. norUDCA choleretic effects may therefore raise safety concerns when used in cholangiopathies with biliary obstruction. We therefore aimed at comparing the effects of UDCA and norUDCA in clear-cut obstructive cholestasis.Methods0.5% UDCA- or norUDCA-fed wild type and Abcb4−/− mice were subjected to CBDL or selective bile duct ligation (SBDL) and compared to controls with regard to liver injury. Bile flow, bile composition, and biliary manometry were compared in UDCA-fed, norUDCA-fed and control mice. Toxicity of UDCA and norUDCA was compared in vitro.ResultsCompared to UDCA, liver injury in CBDL mice was significantly lower in almost all norUDCA groups. In SBDL mice, only UDCA induced bile infarcts in the ligated lobes, whereas norUDCA even ameliorated liver injury. In vitro, UDCA induced cellular ATP depletion and was significantly more toxic than norUDCA in HepG2 cells, mouse bile duct epithelial cells, and primary human hepatocytes.ConclusionsCompared to norUDCA, UDCA is significantly more toxic in CBDL mice. norUDCA, in contrast to UDCA, significantly ameliorates liver injury in SBDL mice. Our findings uncover profound differences in metabolism and therapeutic mechanisms of both bile acids with important clinical consequences.

Translating an Understanding of the Pathogenesis of Hepatic Fibrosis to Novel Therapies

The response to injury is one of wound healing and fibrogenesis, which ultimately leads to fibrosis. The fibrogenic response to injury is a generalized one across virtually all organ systems. In the liver, the injury response, typically occurring over a prolonged period of time, leads to cirrhosis (although it should be pointed out that not all patients with liver injury develop cirrhosis). The fact that many different diseases result in cirrhosis suggests a common pathogenesis. The study of hepatic fibrogenesis over the past 2 decades has been remarkably active, leading to a considerable understanding of this process. It clearly has been shown that the hepatic stellate cell is a central component in the fibrogenic process. It also has been recognized that other effector cells are important in the fibrogenic process, including resident fibroblasts, bone marrow-derived cells, fibrocytes, and even perhaps cells derived from epithelial cells (ie, through epithelial to mesenchymal transition). A key aspect of the biology of fibrogenesis is that the fibrogenic process is dynamic; thus, even advanced fibrosis (or cirrhosis) is reversible. Together, an understanding of the cellular basis for liver fibrogenesis, along with multiple aspects of the basic pathogenesis of fibrosis, have highlighted many exciting potential therapeutic opportunities. Thus, although the most effective antifibrotic therapy is simply treatment of the underlying disease, in situations in which this is not possible, specific antifibrotic therapy is likely not only to become feasible, but will soon become a reality. This review highlights the mechanisms underlying fibrogenesis that may be translated into future antifibrotic therapies and to review the current state of clinical development.

In vitro and in vivo characteristics of hepatic oval cells modified with human hepatocyte growth factor

Hepatocyte growth factor (HGF) is a multifunctional growth factor that controls cell scattering. It has been suggested that it regulates the proliferation of hepatic oval cells (HOCs). Using a HOC line that stably expresses the human HGF gene (hHGF), we investigated the in vitro proliferation and differentiation characteristics of hHGF-modified HOCs and explored their potential capacity for intrahepatic transplantation. A modified 2-acetylaminofluorene and partial hepatectomy (2-AAF/PH) model was established to activate the proliferation of oval cells in the rat liver. HOCs were transfected with the pBLAST2-hHGF plasmid and hHGF-carrying HOCs were selected based on blasticidin resistance. The level of hHGF secretion was determined via ELISA. Cell proliferation was determined using the MTT assay. Differentiation was induced by growth factor withdrawal. A two-cuff technique was used for orthotopic liver transplantation, and HOCs or hHGF-modified HOCs were transplanted into the recipients. The levels of biochemical indicators of liver function were measured after transplantation. An HOC line stably expressing hHGF was established. The transfected line showed greater hHGF secretion than normal HOCs. The hHGF gene promoted the proliferation capability of HOCs by reducing the peak time in vitro. The hHGF-modified HOCs differentiated into hepatocytes and bile duct epithelial cells upon growth factor withdrawal in vitro. In addition, hHGF-modified HOC transplantation significantly prolonged the median survival time (MST) and improved the liver function of recipients compared to HOC transplant recipients and nontransplanted controls. Our results indicate that hHGF-modified HOCs may have valuable properties for therapeutic liver regeneration after orthotopic liver transplantation.

Association analysis of toll-like receptor 4 polymorphisms in Japanese primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is characterized by portal inflammation and immune-mediated destruction of intrahepatic bile ducts that often result in liver failure. Toll-like receptor (TLR) 4 recognizes lipopolysaccharides of Gram-negative bacteria. Infectious agents have been suspected to play a crucial role in PBC pathogenesis since TLR4 expression was found in bile duct epithelial cells and periportal hepatocytes in liver tissues of PBC. To assess the potential contribution of TLR4 SNPs to the development of this disease, we genotyped five SNPs in TLR4 in 261 PBC patients and 359 controls using a TaqMan assay. No significant positive associations with either PBC susceptibility or progression were uncovered. These results indicate that TLR4 polymorphisms do not play a prominent role in the development of PBC in Japanese patients.

Mechanosensitive Cl− secretion in biliary epithelium mediated through TMEM16A

Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Alterations of the SWI/SNF chromatin remodelling subunit‐BRG1 and BRM in hepatocellular carcinoma

The SWI/SNF chromatin remodelling complex, which contains either brahma-related gene-1 (BRG1) or brahma (BRM) as the catalytic ATPase, functions as a master regulator of gene expression. To examine alterations of BRG1 and BRM in hepatocellular carcinoma (HCC). We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumours, and conducted further searches for mutations in BRG1 and BRM genes. Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumours respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumours, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumours, compared to non-tumour tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival. Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear.

The Intimate Relationship between Congenital Hepatic Fibrosis and Autosomal Recessive Polycystic Kidney Disease: A Case Report

Case: This case describes a 38-year-old Korean female with history of hepatitis B (prior positive for Hep B core antibody, treated with Epivir in the post renal transplant setting, now with negative Hep B core antibody and undetectable viral load), thrombocytopenia, diabetes mellitus type II, chronic kidney disease secondary to polycystic kidney disease, status post cadaveric CMV positive renal transplant, with multiple episodes of urosepsis and pyelonephritis. Patient presented with fevers and chills for 1 day with associated nausea and vomiting for 2 days. On admission, the patient denied dysuria or hematuria. Abdominal ultrasound revealed moderately coarse nodular liver parenchyma and multiple cysts in the right liver, the largest one measuring 1.9 × 1.17 cm, and splenomegaly. Patient's family history was positive for polycystic kidney disease in her father. Given the patient's presenting symptoms, radiologic findings, and history of polycystic kidney disease, the patient underwent liver biopsy. Findings were broad fibrous septa with ductal abnormalities suggestive of ductal plate malformation all confirming the diagnosis of Congenital Hepatic Fibrosis. Discussion: Congenital hepatic fibrosis (CHF) is a fibrocystic liver disease that has a close relationship with autosomal recessive polycystic kidney disease (ARPKD). The incidence of ARPKD is around 1:20000, with the incidence of congenital hepatic fibrosis reported to be 43% to 83%. This close relationship has been linked to a mutation in the PKHD1 gene, which encodes a protein found on the primary cilia of renal and bile duct epithelial cells and has been recognized to maintain the tubular architecture of cells in the kidney and liver. ARPKD normally occurs early in patient's life, and will lead to dialysis and/or transplant early in life. Patients who survive past infancy generally seem to have a milder renal phenotype and will maintain good renal function into adulthood, but are at higher risk of liver disease, especially in setting of renal transplantation, which allows patients to live longer. The initial diagnosis is made by identification of an anatomic lesion via ultrasound, CT, MRI and/or MRCP, which then is confirmed by liver biopsy. Recognizing the intimate relationship between ARPKD and CHF and early signs of ensuing portal hypertension are of clinical relevance in early diagnosis and intervention. The goal is to decrease the incidence of bleeding and infection and ultimately to reduce morbidity and mortality in this unique population.Table: Clinical features and complications of congenital hepatic fibrosis

Human hepatocyte growth factor (hHGF)-modified hepatic oval cells improve liver transplant survival.

Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF) in modifying hepatic oval cells (HOCs) administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05). Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) levels while increasing the production of IL-10 and TGF-β1 (P<0.05). HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only). Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver injuries.

α1- and α5-containing Laminins Regulate the Development of Bile Ducts via β1 Integrin Signals

Signals derived from basal lamina components are important for developing three-dimensional architecture of epithelial tissues. Laminins consisting of α, β, and γ subunits in basal lamina play pivotal roles in the formation and maintenance of epithelial tissue structures. However, it remains unclear which laminin isoforms transmit signals and how epithelial cells receive them to regulate multiple developmental processes. In three-dimensional culture of a liver progenitor cell line, Hepatic Progenitor Cells Proliferating on Laminin (HPPL), the cells establish apicobasal polarity and form cysts with a central lumen. Neutralizing antibody against β1 integrin blocked the formation and maintenance of the cyst structure, indicating that β1 integrin signaling was necessary throughout the morphogenesis. Although the addition of α1-containing laminin, a ligand of β1 integrin, induced cyst formation, it was dispensable for the maintenance of the cyst, suggesting that HPPL produces another ligand for β1 integrin to maintain the structure. Indeed, we found that HPPL produced α5-containing laminin, and siRNA against laminin α5 partially inhibited the lumen formation. In fetal liver, p75NTR(+) periportal fibroblasts and bile duct epithelial cells, known as cholangiocytes, expressed α1- and α5-containing laminins, respectively. In laminin α5 KO liver, cholangiocytes normally emerged, but the number of bile ducts was decreased. These results suggest that α1-containing laminin is sufficient as a component of the basal lamina for the commitment of bipotential liver progenitors to cholangiocytes and the apicobasal polarization, whereas α5-containing laminin is necessary for the formation of mature duct structures. Thus, α1- and α5-containing laminins differentially regulate the sequential events to form epithelial tissues via β1 integrin signals.

The cholangiocyte marker, BD. 1, forms a stable complex with CLIP170 and shares an identity with eIF3a, a multifunctional subunit of the eIF3 initiation complex

We have previously described the generation of a monoclonal antibody recognizing a novel cholangiocyte marker, designated BD.1, that is expressed by fetal and adult rat cholangiocytes but not hepatocytes or the hepatic progenitor cells known as oval cells. In the present report, we have undertaken a comprehensive examination of BD.1 expressed by long-term cultures of bile duct epithelial cells (BDEC) and prostate epithelial cells (PEC). We show that with continued passage, the levels of BD.1 expressed by BDEC and PEC drop significantly, a decrease that is temporally associated with transition from a diploid to an aneuploid karyotype. Cell cycle analysis revealed cell cycle dependent expression of BD.1 characterized by decreased BD.1 levels within the first 10h after release from serum starvation followed by reacquisition as cells entered S phase. MAb BD.1 recognized a 170kDa protein in Western blots and showed strong reactivity with a 170kDa band in blots prepared from phosphoproteins isolated by metal affinity chromatography. Analysis by mass spectrometry of tryptic peptides generated from BD.1 purified by continuous elution electrophoresis identified the plus end microtubule‐binding protein, CLIP170, in the fraction reactive with MAb BD.1. Double immunofluorescence with MAb BD.1 and a MAb specific for CLIP170 showed that both were reactive with intrahepatic bile ducts. However, overexpression or siRNA knockdown of CLIP170 in 293T cells did not significantly alter BD.1 levels, indicating that CLIP170 and BD.1 were distinct, co-migrating proteins. Immunoprecipitation analysis with MAb BD.1 and anti-CLIP170 antibodies showed that under microtubule depolymerizing conditions the two proteins could be co-precipitated with both antibodies, leading us to conclude they were capable of forming stable complexes. Two different protocols were devised to enrich for the CLIP170 binding protein recognized by MAb BD.1. Analysis of tryptic peptides by LC-ESI-MS/MS identified BD.1 as eIF3a, the largest subunit of the elongation initiation factor 3 (eIF3) complex. This identity was confirmed by the simultaneous knockdown of both BD.1 and eIF3a by eIF3a-specific siRNAs and by the strong reactivity of MAb BD.1 with the 170kDa protein immunoprecipitated with the anti-eIF3a antibody, 5H10. Based on these findings, we concluded that the BD.1 antigen was identical to eIF3a, a multifunctional subunit of the eIf3 complex shown here to associate with microtubules through its interactions with CLIP170.

Study on hepatotoxicity of aqueous extracts of Polygonum multiflorum in rats after 28-day oral administration-analysis on correlation of cholestasis

To observe the liver injury degree of SD rats after 28-day administration of aqueous extracts of Polygonum multiflorum (AEPM) and the correlation with cholestasis mechanism. Adult SD rats were orally administered with 30, 60 g x kg(-1) of APEM once every day for 28 d. After 28 d, the general condition of rats such as weight were observed, liver function-related indicators were detected. Bile was collected to determine total bile acid output, flow rate and density and changes in major compositions. Their livers were weighed then sent for histopathological examination. AEPM did not change the general conditions and weights of rats. From the results of the related indicators of liver function and cholestasis, AEPM did not change the contents of ALT and AST in serum, but high dose of AEPM can increase the contents of ALP, GGT and TBA in serum (P < 0.05, P < 0.01) and decrease the content of TBIL in serum (P < 0.05). And the contents of GGT in serum of low dose rats were increased (P < 0.05). The bile flow was not changed by AEPM, but bile compositions of high dose male rats were obviously changed (TG increase, TBIL decrease, TBA decrease). The weights of liver and ratio of liver of the high dose rats were increased but showed no statistical significance. Pathologic examination displayed that there were only small pieces of necrosis in livers of several rats, without any severe disease. AEPM can obviously injure bile duct epithelial cells, intervene liver cell functions and change bile compositions in rats, thus it is proved to induce cholestasis without severe liver injury.

Lipopolysaccharide enhances transforming growth factor β1‐induced platelet‐derived growth factor‐B expression in bile duct epithelial cells

Platelet-derived growth factor (PDGF)-B is a potent profibrogenic mediator expressed by bile duct epithelial cells (BDECs) that contributes to liver fibrosis after bile duct ligation. However, the mechanism of PDGF-B induction in BDECs during cholestasis is not known. Transforming growth factor β (TGFβ) and lipopolysaccharide (LPS) also contribute to the profibrogenic response after bile duct ligation. We tested the hypothesis that LPS and TGFβ1 synergistically induce PDGF-B expression in BDECs. Transformed human BDECs (MMNK-1 cells) and primary rat BDECs were stimulated with LPS and/or TGFβ1, and signaling pathways through which LPS potentiates TGFβ1-induced PDGF-B mRNA expression were investigated. Stimulation of MMNK-1 cells with LPS alone did not significantly induce PDGF-B mRNA expression. However, LPS co-treatment enhanced TGFβ1 induction of PDGF-B mRNA in MMNK-1 cells and also in primary rat BDECs. Importantly, co-treatment of MMNK-1 cells with LPS and TGFβ1 also significantly increased PDGF-BB protein expression. Interestingly, LPS did not affect TGFβ1 activation of a SMAD-dependent reporter construct. Rather, stimulation of MMNK-1 cells with LPS, but not TGFβ1, increased JNK1/2 phosphorylation. Expression of dominant negative JNK2, but not dominant negative JNK1, inhibited the LPS potentiation of TGFβ1-induced PDGF-B mRNA expression in MMNK-1 cells. In addition, LPS treatment caused IκBα degradation and activation of a nuclear factor κB (NFκB)-dependent reporter construct. Expression of an IκBα super repressor inhibited activation of NFκB and attenuated LPS potentiation of TGFβ1-induced PDGF-B mRNA. The results indicate that LPS activation of NFκB and JNK2 enhances TGFβ1-induced PDGF-B expression in BDECs.

Feeder-Free and Serum-Free Production of Hepatocytes, Cholangiocytes, and Their Proliferating Progenitors from Human Pluripotent Stem Cells: Application to Liver-Specific Functional and Cytotoxic Assays

We have established a serum- and feeder-free culture system for the efficient differentiation of multifunctional hepatocytes from human embryonic stem (ES) cells and three entirely different induced pluripotent stem (iPS) cells (including vector/transgene-free iPS cells generated using Sendai virus vector) without cell sorting and gene manipulation. The differentiation-inducing protocol consisted of a first stage; endoderm induction, second stage; hepatic initiation, and third stage; hepatic maturation. At the end of differentiation culture, hepatocytes induced from human pluripotent stem cells expressed hepatocyte-specific proteins, such as α-fetoprotein, albumin, α1 antitrypsin and cytochrome P450 (CYP3A4), at similar or higher levels compared with three control human hepatocyte or hepatic cell lines. These human iPS/ES cell-derived hepatocytes also showed mature hepatocyte functions: indocyanine green dye uptake (≈ 30%), storage of glycogen (>80%) and metabolic activity of CYP3A4. Furthermore, they produced a highly sensitive hepatotoxicity assay system for D-galactosamine as determined by the extracellular release of hepatocyte-specific enzymes. Hepatoprotective prostaglandin E1 attenuated this toxicity. Interestingly, bile duct-specific enzymes were also detected after drug treatment, suggesting the presence of bile-duct epithelial cells (cholangiocytes) in our culture system. Electron microscopic studies confirmed the existence of cholangiocytes, and an immunostaining study proved the presence of bipotential hepatoblasts with high potential for proliferation. Differentiated cells were transferrable onto new dishes, on which small-sized proliferating cells with hepatocyte markers emerged and expanded. Thus, our differentiation culture system provides mature functional hepatocytes, cholangiocytes, and their progenitors with proliferative potential from a wide variety of human pluripotent stem cells.

Cytokine gene expression in the livers of ducklings infected with duck hepatitis virus-1 JX strain

Duck hepatitis virus type 1 (DHV-1) causes a highly contagious disease in ducklings and is often associated with liver necrosis, hemorrhages, and high mortality. In the current study, the expression levels of gene transcripts encoding proinflammatory cytokines and the virus were measured by quantitative reverse-transcription PCR in duck livers after infection with a DHV-1 JX isolate obtained from natural cases in Hubei Province, China. In addition, sera IL-1β, IL-6, and alanine aminotransferase levels were quantified. Liver histopathology was examined following DHV-1 infection. The ducklings died within 1 to 2 d postinfection (d.p.i.) because of typical liver degeneration, hemorrhage, necrosis, and bile-duct epithelial cell proliferation. Transcripts of the cytokines IFN-α, IL-6, TNF-α, and IL-10 decreased by 0.5 d.p.i. and then gradually increased at 1 d.p.i. Similarly, DHV-1 JX 3D gene levels in the liver sharply increased at 1 d.p.i. and then maintained a high level. In contrast, liver TNF-α and IL-1β transcripts showed no increased expression of the cytokine gene postinfection and significantly decreased compared with the expression at 0.25 d.p.i., only the expression of IFN-α transcripts increased 128-fold by 1 d.p.i. Changes in the serum IL-6 level remained relatively stable postinfection and not significantly different compared with that of the control (P > 0.05), whereas serum levels of IL-1β significantly decreased at 0.5 d.p.i. and increased from 1 d.p.i. onwards (P < 0.05). Serum alanine aminotransferase levels significantly increased 2 d.p.i. compared with that of the control group (P < 0.01), which seemed to keep with the number of dead ducks. The cytokines exhibited a biphasic pattern following DHV-1 JX infection. Taken together, the data indicated that duckling liver inflammatory responses were produced following experimental DHV-1 JX infection involving multiple cytokines.

Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig

Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.

Transplanted Fibroblast Cell Sheets Promote Migration of Hepatic Progenitor Cells in the Incised Host Liver in Allogeneic Rat Model

Introduction: ‘Cell-sheet engineering’ using temperature-responsive dishes has been noted as a new approach in the tissue engineering field. This technique has been already used in clinical fields including autologous mucosal epithelial cell sheets for the treatment of esophageal ulceration after endoscopic submucosal dissection. Usefulness of cell-sheet engineering has been expected also in hepatic tissue engineering. on the other hand, it was reported that proliferation of smooth muscle cells in the site where skeletal cell sheets were transplanted successfully prevented the deterioration of the impaired myocardium. These data indicate that transplanted cell sheets may induce organ-specific progenitor cells. The objective of this study is to explore procedure to induce hepatic progenitor cells in the rat liver. Methods: SD rat dermal fibroblasts (DFs) were used as donor cells and male F344 nude rats as recipients. DFs were cultured on temperature-responsive dishes and DFs were then harvested as a cell sheet. In the DF group, DFs sheets were transplanted into the incised surface of the liver. In the control group, only a same incision was made. The rats were sacrificed on postoperative days (POD) 7, 14, and 28 (n=5). for histological examination, hematoxyline-eosin staining and immunohistochemistry for cytokeratin (CK)-8 (rat bile duct epithelial cell marker), OV-6 (liver progenitor cell marker) and α-fetoprotein (AFP) (immature hepatocytemarker) were performed. We also performed the transplantation of GFP-expressing DFs sheet into the incised surface of the liver. Results: In the control group, necrosis or inflammatory cell infiltration was observed on POD7, and those were absorbed eventually. in contrast, the DF sheet transplanted sites became thicker, and bile duct (BD)-like structures appeared on POD 7. On POD 14, the BD-like structures were more significant, and immature hepatocyte-like cells were observed. Histological assessment revealed that BD-like structures were CK8 positive, and hepatocyte-like cells induced around the BD were both OV-6 and AFP positive. Observing the GFP-expressing DFs sheet transplanted sites byimmunofluorescent microscope, the proliferation of GFP positive DF cells were found, but the cells forming new structures including BD were GFP negative. These findings may become suggest that engrafted DFs recruited hepatic progenitor cells and supported their differentiation to mature hepatic tissues. Conclusions: We observed engineering process of hepatic tissues after transplantation of DF sheets. We also demonstrated that the origin of newly formed structures was recipient liver, which means transplanted DFs sheets induced hepatic progenitor cells from the recipient liver. The present study described a novel aspect of hepatic differentiation process induced at liver injury site. These technologies may become a useful therapeutic method for liver diseases in the future.

Effects of warm ischemia time on biliary injury in rat liver transplantation

To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats. Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion. With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened. A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.