RESUMEN
Liver transplantation is the only curative option for many liver diseases that end up in liver failure, and cholangiopathy remains a challenging complication post-liver transplant, associated with significant morbidity and potential graft loss. The low availability of organs and high demand for transplantation motivate scientists to find novel interventions. Organoids, as three-dimensional cell cultures derived from adult cells or induced pluripotent cells, may help to address this problem. Different types of organoids have been described, from which cholangiocyte organoids offer a high level of versatility and plasticity for a deeper study of liver disease mechanisms. Cholangiocytes can be obtained from different segments of the biliary tree and have shown a remarkable capacity to adapt to new environments, presenting an effective system for studying cholangiopathies. Studies using cholangiocyte organoids show promising results for disease modeling, where organoids offer fundamental features to recapitulate the complexities of tissues in vitro and uncover fundamental pathological pathways to potentially reveal therapeutic strategies for personalized medicine. Organoids could hold the potential for regeneration of injured livers, representing tools of clinical impact in regenerative medicine when tissue damage is already present.
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Trasplante de Hígado , Organoides , Humanos , Trasplante de Hígado/efectos adversos , Animales , Conductos Biliares/citología , Hígado/citología , Hígado/patología , Células Madre Pluripotentes Inducidas/citología , Medicina Regenerativa/métodos , Hepatopatías/cirugía , Hepatopatías/terapia , Hepatopatías/patologíaRESUMEN
Cholangiocytes, the epithelial cells that line the biliary tree, can proliferate under the stimulation of several factors through both autocrine and paracrine pathways. The cocaine-amphetamine-regulated-transcript (CART) peptide has several physiological functions, and it is widely expressed in several organs. CART increases the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor (BDNF), whose expression has been correlated to the proliferation rate of cholangiocytes. In the present study, we aimed to evaluate the expression of CART and its role in modulating cholangiocyte proliferation in healthy and bile duct ligated (BDL) rats in vivo, as well as in cultured normal rat cholangiocytes (NRC) in vitro. Liver samples from both healthy and BDL (1 week) rats, were analyzed by immunohistochemistry and immunofluorescence for CART, CK19, TrkB and p75NTR BDNF receptors. PCNA staining was used to evaluate the proliferation of the cholangiocytes, whereas TUNEL assay was used to evaluate biliary apoptosis. NRC treated or not with CART were used to confirm the role of CART on cholangiocytes proliferation and the secretion of BDNF. Cholangiocytes proliferation, apoptosis, CART and TrkB expression were increased in BDL rats, compared to control rats. We found a higher expression of TrkB and p75NTR, which could be correlated with the proliferation rate of biliary tree during BDL. The in vitro study demonstrated increased BDNF secretion by NRC after treatment with CART compared with control cells. As previously reported, proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors, including neurotrophins. Accordingly, CART may play a key role in the remodeling of biliary epithelium during cholestasis by modulating the secretion of BDNF.
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Conductos Biliares , Factor Neurotrófico Derivado del Encéfalo , Proteínas del Tejido Nervioso , Animales , Ratas , Conductos Biliares/citología , Conductos Biliares/metabolismo , Conductos Biliares/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proliferación Celular , Epitelio/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
BACKGROUND AND AIMS: Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. APPROACH AND RESULTS: We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, ß-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, ß-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. CONCLUSIONS: Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.
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Conductos Biliares/patología , Atresia Biliar/patología , Colestasis/patología , Células Epiteliales/patología , Organoides/patología , Adolescente , Conductos Biliares/citología , Conductos Biliares/crecimiento & desarrollo , Atresia Biliar/complicaciones , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Niño , Preescolar , Colestasis/etiología , Células Epiteliales/citología , Voluntarios Sanos , Humanos , Lactante , Recién Nacido , Cultivo Primario de Células , Uniones Estrechas/patologíaRESUMEN
Biliary Atresia is a devastating pediatric cholangiopathy affecting the bile ducts of the liver. In this review, we describe recent progress in the understanding of liver development with a focus on cholangiocyte differentiation and how use of technical platforms, including rodent, zebrafish and organoid models, advances our understanding of Biliary Atresia. This is followed by a description of potential pathomechanisms, such as autoimmune responses, inflammation, disturbed apical-basal cell polarity, primary cilia dysfunction as well as beta-amyloid accumulation. Finally, we describe current and emerging diagnostic opportunities and recent translation breakthroughs for Biliary Atresia in the area of emerging therapy development, including immunomodulation and organoid-based systems for liver and bile duct repair.
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Conductos Biliares/citología , Atresia Biliar/diagnóstico , Organoides/patología , Animales , Atresia Biliar/patología , Diferenciación Celular , Modelos Animales de Enfermedad , Células Epiteliales/citología , HumanosRESUMEN
BACKGROUND: LIN28B is functionally driving malignant transformation and relevance to the worse disease outcomes by promoting cancer aggressiveness. However, a typical role of LIN28B in cholangiocarcinoma (CCA) is primarily unknown. In this study, the tumorigenic potential of LIN28B in the cholangiocyte context was investigated. METHODS: Stable LIN28B expression in MMNK-1 cells was generated by infecting with retrovirus-containing LIN28B gene. LIN28B-overexpressing cells were further validated the amount of released cytokines by using human cytokine arrays. After treatment of chemo-drugs, cell viability was subsequently measured using MTT assay. Aldehyde dehydrogenase (ALDH) activity was determined using ALDEFLUOR assay Kit and analyzed by flow cytometry. The mRNA and protein expression levels were respectively assayed by RT-qPCR and western blot. RESULTS: Cytokine release results showed that numerous inflammatory cytokines-chemokines related to cancer initiation and development, such as IL-8, IL-6, VEGF, MCP1, TNF-α were significantly increased in LIN28B-overexpressing MMNK-1 cells. Drug sensitivity test showed that LIN28B-overexpressing MMNK-1 treated cells had a high percentage of cell viability compared to MMNK-1-control treated cells. Activity and expression level of a cancer stem cell marker, ALDH was significantly elevated in LIN28B-overexpressing MMNK-1 cells. Moreover, the activation of an oncogenic signaling pathway, signal transducer and activator of transcription 3 (STAT3) was enhanced in LIN28B-overexpressing MMNK-1 cells. Whereas, growth capacity of LIN28B-overexpressing MMNK-1 cells was found to be reduced in STAT3 inhibition. CONCLUSION: LIN28B can regulate the inflammatory response and resistance to chemotherapy of cholangiocytes through modulation of STAT3 signaling pathway.A recent study suggests that activated cholangiocytes can be induced by regulation of LIN28B/STAT3 pathway and this may partially contribute to the initiating CCA. Here, LIN28B and its downstream signaling could be considered as an attractive therapeutic target in patients with CCA.
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Neoplasias de los Conductos Biliares/genética , Colangiocarcinoma/genética , Resistencia a Antineoplásicos/genética , Proteínas de Unión al ARN/fisiología , Factor de Transcripción STAT3/metabolismo , Conductos Biliares/citología , Línea Celular Tumoral , Supervivencia Celular/genética , Citocinas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Oncogenes/genética , Transducción de Señal/genéticaRESUMEN
We previously identified an up-regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up-regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b-flox mice were bred with Krt19-cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin-Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild-type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury.
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Conductos Biliares/citología , Proliferación Celular/genética , Colestasis/genética , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Wnt/deficiencia , Animales , Ácidos y Sales Biliares/metabolismo , Senescencia Celular/genética , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Fatty liver diseases, such as non-alcoholic fatty liver disease (NAFLD), are global health disparities, particularly in the United States, as a result of cultural eating habits and lifestyle. Pathological studies on NAFLD have been mostly focused on hepatocytes and other inflammatory cell types; however, the impact of other biliary epithelial cells (i.e., cholangiocytes) in the promotion of NAFLD is growing. This review article will discuss how cholestatic injury and cholangiocyte activity/ductular reaction influence NAFLD progression. Furthermore, this review will provide informative details regarding the fundamental properties of cholangiocytes and bile acid signaling that can influence NAFLD. Lastly, studies relating to the pathogenesis of NAFLD, cholangiopathies, and ductular reaction will be analyzed to help gain insight for potential therapies.
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Conductos Biliares/metabolismo , Colestasis/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Transducción de Señal , Ácidos y Sales Biliares/metabolismo , Conductos Biliares/citología , Cannabinoides/metabolismo , Colestasis/etiología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Sistemas Neurosecretores/metabolismo , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Cholestatic liver diseases including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are associated with active hepatic fibrogenesis, which can ultimately lead to the development of cirrhosis. However, the exact relationship between the development of liver fibrosis and the progression of cholestatic liver disease remains elusive. Periductular fibroblasts located around the bile ducts seem biologically different from hepatic stellate cells (HSCs). The fibrotic events in these clinical conditions appear to be related to complex crosstalk between immune/inflammatory mechanisms, cytokine signalling, and perturbed homeostasis between cholangiocytes and mesenchymal cells. Several animal models including bile duct ligation (BDL) and the Mdr2-knockout mice have improved our understanding of mechanisms underlying chronic cholestasis. In the present review, we aim to elucidate the mechanisms of fibrosis in order to help to identify potential diagnostic and therapeutic targets.
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Colestasis Intrahepática/metabolismo , Transducción de Señal , Animales , Conductos Biliares/citología , Conductos Biliares/metabolismo , Conductos Biliares/patología , Colestasis Intrahepática/patología , Fibrosis , Humanos , Hígado/citología , Hígado/metabolismo , Hígado/patologíaRESUMEN
BACKGROUND AND AIMS: Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease. We found microRNA-34a (miR-34a), as the downstream gene of p53, was overexpressed in some of fibrogenic diseases. In this study, we sought to explore whether miR-34a plays a role in the fibrosis of PBC. METHODS: The peripheral blood of PBC patients and controls was collected to analyze the level of miR-34a. Human intrahepatic biliary epithelial cells (HIBEC) were cultured. The expression of miR-34a was regulated by miR-34a mimics and inhibitor. The biomarkers of epithelium-mesenchymal transition (EMT), fibrogenesis, inflammation, and transforming growth factor- (TGF-) ß1/smad pathway were analyzed. RESULTS: We found that miR-34a was overexpressed in the peripheral blood in PBC patients. In vitro, overexpressed miR-34a increased the EMT and fibrogenesis activity of HIBEC. Transforming growth factor-beta type 1 receptor (TßR1), TGF-ß1, and p-smad2/3 were upregulated by miR-34a. Inflammatory factors such as IL-6 and IL-17 were also upregulated. Finally, we showed that miR-34a promoted EMT and liver fibrosis in PBC by targeting the TGF-ß1/smad pathway antagonist transforming growth factor-beta-induced factor homeobox 2 (TGIF2). CONCLUSIONS: Our findings show that miR-34a plays an important role in the EMT and fibrosis of PBC through the TGF-ß1/smad pathway by targeting TGIF2. This study suggests that miR-34a may be a new marker of fibrogenesis in PBC. Inhibition of miR-34a may be a promising strategy in treating PBC and improving the prognosis of the disease.
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Transición Epitelial-Mesenquimal/genética , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática/genética , MicroARNs/metabolismo , Conductos Biliares/citología , Conductos Biliares/patología , Estudios de Casos y Controles , Células Epiteliales , Transición Epitelial-Mesenquimal/efectos de los fármacos , Células HEK293 , Humanos , Hígado/inmunología , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/inmunología , Cirrosis Hepática/patología , Cirrosis Hepática Biliar/sangre , Cirrosis Hepática Biliar/genética , Cirrosis Hepática Biliar/inmunología , MicroARNs/agonistas , MicroARNs/antagonistas & inhibidores , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND AND AIMS: Human NAFLD is characterized at early stages by hepatic steatosis, which may progress to NASH when the liver displays microvesicular steatosis, lobular inflammation, and pericellular fibrosis. The secretin (SCT)/secretin receptor (SCTR) axis promotes biliary senescence and liver fibrosis in cholestatic models through down-regulation of miR-125b signaling. We aim to evaluate the effect of disrupting biliary SCT/SCTR/miR-125b signaling on hepatic steatosis, biliary senescence, and liver fibrosis in NAFLD/NASH. APPROACH AND RESULTS: In vivo, 4-week-old male wild-type, Sct-/- and Sctr-/- mice were fed a control diet or high-fat diet (HFD) for 16 weeks. The expression of SCT/SCTR/miR-125b axis was measured in human NAFLD/NASH liver samples and HFD mouse livers by immunohistochemistry and quantitative PCR. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were evaluated in mouse liver and human NAFLD/NASH liver samples. miR-125b target lipogenesis genes in hepatocytes were screened and validated by custom RT2 Profiler PCR array and luciferase assay. Biliary SCT/SCTR expression was increased in human NAFLD/NASH samples and in livers of HFD mice, whereas the expression of miR-125b was decreased. Biliary/hepatocyte senescence, ductular reaction, and liver angiogenesis were observed in human NAFLD/NASH samples as well as HFD mice, which were decreased in Sct-/- and Sctr-/- HFD mice. Elovl1 is a lipogenesis gene targeted by miR-125b, and its expression was also decreased in HFD mouse hepatocytes following Sct or Sctr knockout. Bile acid profile in fecal samples have the greatest changes between wild-type mice and Sct-/- /Sctr-/- mice. CONCLUSION: The biliary SCT/SCTR/miR-125b axis promotes liver steatosis by up-regulating lipid biosynthesis gene Elovl1. Targeting the biliary SCT/SCTR/miR-125b axis may be key for ameliorating phenotypes of human NAFLD/NASH.
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Enfermedad del Hígado Graso no Alcohólico/genética , Receptores Acoplados a Proteínas G/genética , Receptores de la Hormona Gastrointestinal/genética , Secretina/genética , Animales , Conductos Biliares/citología , Conductos Biliares/metabolismo , Línea Celular , Senescencia Celular/genética , Modelos Animales de Enfermedad , Elongasas de Ácidos Grasos/genética , Elongasas de Ácidos Grasos/metabolismo , Ácidos Grasos no Esterificados , Hepatocitos/metabolismo , Humanos , Lipogénesis/genética , Ratones , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fenotipo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de la Hormona Gastrointestinal/metabolismo , Secretina/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Biliary tract infection with the carcinogenic human liver fluke, Clonorchis sinensis, provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma. Complications are proportional to the intensity and duration of the infection. In addition to mechanical irritation of the biliary epithelia from worms, their excretory-secretory products (ESPs) cause chemical irritation, which leads to inflammation, proliferation, and free radical generation. METHODS: A three-dimensional in vitro cholangiocyte spheroid culture model was established, followed by ESP treatment. This allowed us to examine the intrinsic pathological mechanisms of clonorchiasis via the imitation of prolonged and repetitive in vivo infection. RESULTS: Microarray and RNA-Seq analysis revealed that ESP-treated cholangiocyte H69 spheroids displayed global changes in gene expression compared to untreated spheroids. In ESP-treated H69 spheroids, 185 and 63 probes were found to be significantly upregulated and downregulated, respectively, corresponding to 209 genes (p < 0.01, fold change > 2). RNA-Seq was performed for the validation of the microarray results, and the gene expression patterns in both transcriptome platforms were well matched for 209 significant genes. Gene ontology analysis demonstrated that differentially expressed genes were mainly classified into immune system processes, the extracellular region, and the extracellular matrix. Among the upregulated genes, four genes (XAF1, TRIM22, CXCL10, and BST2) were selected for confirmation using quantitative RT-PCR, resulting in 100% similar expression patterns in microarray and RNA-Seq. CONCLUSIONS: These findings broaden our understanding of the pathological pathways of liver fluke-associated hepatobiliary disorders and suggest a novel therapeutic strategy for this infectious cancer.
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Conductos Biliares/parasitología , Clonorquiasis/genética , Clonorchis sinensis/metabolismo , Proteínas del Helminto/metabolismo , Esferoides Celulares/parasitología , Animales , Conductos Biliares/citología , Clonorquiasis/metabolismo , Clonorquiasis/parasitología , Clonorchis sinensis/genética , Células Epiteliales/metabolismo , Células Epiteliales/parasitología , Perfilación de la Expresión Génica , Proteínas del Helminto/genética , Humanos , Masculino , Conejos , Esferoides Celulares/citología , Esferoides Celulares/metabolismoRESUMEN
BACKGROUND: Liver fibrosis is defined as the accumulation of the extracellular matrix and scar formation. The receptor for advanced glycation end products (RAGE) has been demonstrated to participate in fibrogenesis. S100B is a ligand of RAGE and exerts extracellular functions by inducing a series of signal transduction cascades. However, the involvement of S100B and RAGE in cholestasis-induced liver fibrosis remains unclear. In this study, we investigated S100B and RAGE expression during liver fibrosis in mice that underwent common bile duct ligation (BDL). METHODS: BDL was performed in 10-week-old male C57BL/6J mice with sham control (n = 26) and BDL (n = 26) groups. Expression levels of S100B, RAGE and fibrotic markers in the livers from both groups at week 1 and 3 after BDL were examined by western blot and quantitative real-time reverse transcription polymerase chain reaction analysis. Liver fibrotic changes were examined by histological and ultrastructural analysis. RESULTS: Histological staining with Sirius Red and the evaluation of the messenger RNA expression of fibrotic markers showed noticeable periportal fibrosis and bile duct proliferation. S100B was mainly present in bile duct epithelial cells, and its expression was upregulated in proportion to the ductular reaction during fibrogenesis by BDL. RAGE expression was also increased, and interestingly, triple immunofluorescence staining and transmission electron microscopy showed that both S100B and RAGE were expressed in proliferating bile duct epithelial cells and activated hepatic stellate cells (HSCs) of the BDL livers. In addition, in rat HSCs (HSC-T6), treatment with recombinant S100B protein significantly increased fibrotic markers in a dose-dependent manner, and RAGE small interfering RNA (siRNA) suppressed S100B-stimulated upregulation of fibrotic markers compared with cells treated with scramble siRNA and S100B. CONCLUSION: These findings suggest that the increased expression of S100B and RAGE and the interaction between S100B and RAGE may play an important role in ductular reaction and liver fibrosis induced by BDL.
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Cirrosis Hepática/patología , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Subunidad beta de la Proteína de Unión al Calcio S100/metabolismo , Animales , Conductos Biliares/citología , Conductos Biliares/cirugía , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/metabolismo , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Receptor para Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Receptor para Productos Finales de Glicación Avanzada/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Subunidad beta de la Proteína de Unión al Calcio S100/genética , Subunidad beta de la Proteína de Unión al Calcio S100/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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.
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Enfermedades de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos/fisiología , Conductos Biliares/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Epiteliales/citología , Organoides/trasplante , Animales , Bilis , Conductos Biliares/fisiología , Conductos Biliares Intrahepáticos/citología , Conducto Colédoco/citología , Células Epiteliales/fisiología , Vesícula Biliar/citología , Regulación de la Expresión Génica , Humanos , Hígado/fisiología , Trasplante de Hígado , Trasplante de Células Madre Mesenquimatosas , Ratones , Organoides/fisiología , RNA-Seq , Obtención de Tejidos y Órganos , TranscriptomaRESUMEN
Hedgehog morphogens govern multiple aspects of pancreas organogenesis and functioning with diverse outcomes across species. Although most current differentiation protocols repress Sonic hedgehog (SHH) signals during in vitro endocrine specification, the role and mechanisms through which the SHH pathway antagonizes pancreas development during in vitro human embryonic stem (hES) cell differentiation remain unclear. We modulated SHH signaling at transitory stages of hES cell-derived pancreatic progenitors and analyzed the effect on cellular fate decisions. We identify the Hedgehog pathway as a negative regulator of pancreatic endoderm formation through up-regulation of a set of pancreatobiliary markers required for ductal specification, including SOX17, FOXA2, HNF1ß, HNF6, PDX1, and SOX9. Surprisingly, active Hedgehog signals impeded a group of pancreatic epithelium markers, including HNF4α, HHEX, PAX6, and PTF1α. To understand how SHH signals repress the transcription of these specific markers, we analyzed Polycomb group proteins. We found differential expression of Polycomb Repressive Complex 1 subunit, BMI1 upon Shh pathway modulation in the pancreatic progenitors. Ectopic activation of Sonic hedgehog results in over-expression of BMI1 and its associated repressive histone mark, H2AK119Ub1, in the multipotent progenitors. Our data suggest that Sonic hedgehog restricts the pancreatic differentiation program by limiting progenitor cells acquiring pancreatic epithelial fates and instead promotes pancreatobiliary differentiation. We further provide mechanistic cues of an association between Hedgehog signaling and epigenetic silencers during pancreatic lineage decisions.
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Endodermo/embriología , Redes Reguladoras de Genes , Proteínas Hedgehog/metabolismo , Células Madre Embrionarias Humanas/citología , Páncreas/embriología , Transducción de Señal , Conductos Biliares/citología , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Humanos , Modelos Biológicos , Proteínas del Grupo Polycomb/metabolismo , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field. Here, we describe a protocol for the continuous patterning of hepatic, biliary and pancreatic (HBP) structures from a 3D culture of human pluripotent stem cells (PSCs). After differentiating PSCs into anterior and posterior gut spheroids, the two spheroids are fused together in one well. Subsequently, self-patterning of multi-organ (i.e., HBP) domains occurs within the boundary region of the two spheroids, even in the absence of any extrinsic factors. Long-term culture of HBP structures induces differentiation of the domains into segregated organs complete with developmentally relevant invagination and epithelial branching. This in-a-dish model of human hepato-biliary-pancreatic organogenesis provides a unique platform for studying human development, congenital disorders, drug development and therapeutic transplantation. More broadly, our approach could potentially be used to establish inter-organ connectivity models for other organ systems derived from stem cell cultures.
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Técnicas de Cultivo de Célula/métodos , Organoides/citología , Ingeniería de Tejidos/métodos , Conductos Biliares/citología , Diferenciación Celular/fisiología , Humanos , Hígado/citología , Organogénesis/fisiología , Organoides/metabolismo , Páncreas/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.
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Plasticidad de la Célula/genética , Regeneración Hepática/genética , Hígado/fisiología , Animales , Conductos Biliares/citología , Diferenciación Celular/genética , Epigénesis Genética , Células Epiteliales/fisiología , Hepatocitos/fisiología , Hepatocitos/trasplante , Humanos , Hígado/citología , Masculino , Ratones , Ratones Transgénicos , RNA-Seq , Análisis de la Célula Individual , Proteína Smad4/genéticaRESUMEN
BACKGROUND & AIMS: Transforming growth factor ß (TGFß) upregulates cholangiocyte-derived signals that activate myofibroblasts and promote fibrosis. Using epigenomic and transcriptomic approaches, we sought to distinguish the epigenetic activation mechanisms downstream of TGFß that mediate transcription of fibrogenic signals. METHODS: Chromatin immunoprecipitation (ChIP)-seq and RNA-seq were performed to assess histone modifications and transcriptional changes following TGFß stimulation. Histone modifications and acetyltransferase occupancy were confirmed using ChIP assays. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) was used to investigate changes in chromatin accessibility. Cholangiocyte cell lines and primary cholangiocytes were used for in vitro studies. Mdr2-/- and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC)-fed mice were used as animal models. RESULTS: TGFß stimulation caused widespread changes in histone 3 lysine 27 acetylation (H3K27ac), and was associated with global TGFß-mediated transcription. In contrast, H3K9ac was gained in a smaller group of chromatin sites and was associated with fibrosis pathways. These pathways included overexpression of hepatic stellate cell (HSC) activators such as fibronectin 1 (FN1) and SERPINE1. The promoters of these genes showed H3K9ac enrichment following TGFß. Of the acetyltransferases responsible for H3K9ac, cholangiocytes predominantly express Lysine Acetyltransferases 2A (KAT2A). Small interfering RNA knockdown of KAT2A or H3K9ac inhibition prevented the TGFß-mediated increase in FN1 and SERPINE1. SMAD3 ChIP-seq and ATAC-seq suggested that TGFß-mediated H3K9ac occurs through SMAD signaling, which was confirmed using colocalization and genetic knockdown studies. Pharmacologic inhibition or cholangiocyte-selective deletion of Kat2a was protective in mouse models of biliary fibrosis. CONCLUSIONS: Cholangiocyte expression of HSC-activating signals occurs through SMAD-dependent, KAT2A-mediated, H3K9ac, and can be targeted to prevent biliary fibrosis.
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Conductos Biliares/patología , Epigénesis Genética/genética , Histonas/metabolismo , Cirrosis Hepática Biliar/genética , Factor de Crecimiento Transformador beta/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Acetilación/efectos de los fármacos , Animales , Conductos Biliares/citología , Conductos Biliares/efectos de los fármacos , Línea Celular , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Epigénesis Genética/efectos de los fármacos , Epigenómica , Técnicas de Silenciamiento del Gen , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Cirrosis Hepática Biliar/inducido químicamente , Cirrosis Hepática Biliar/tratamiento farmacológico , Cirrosis Hepática Biliar/patología , Ratones , Ratones Noqueados , Miofibroblastos/patología , Cultivo Primario de Células , Piridinas/administración & dosificación , Piridinas/toxicidad , RNA-Seq , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.
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Conductos Biliares/metabolismo , Hepatocitos/metabolismo , Esferoides Celulares/metabolismo , Ingeniería de Tejidos , Animales , Conductos Biliares/citología , Células Cultivadas , Hepatocitos/citología , Hígado , Ratas , Esferoides Celulares/citologíaRESUMEN
BACKGROUND & OBJECTIVES: Alcoholic hepatitis (AH) is a common but life-threatening disease with limited treatment options. It is thought to result from hepatocellular damage, but the presence of cholestasis worsens prognosis, so we examined whether bile ducts participate in the pathogenesis of this disease. DESIGN: Cholangiocytes derived from human bile ducts were co-cultured with neutrophils from patients with AH or controls. Loss of type 3 inositol 1,4,5-trisphosphate receptor (ITPR3), an apical intracellular calcium channel necessary for cholangiocyte secretion, was used to reflect cholestatic changes. Neutrophils in contact with bile ducts were quantified in liver biopsies from patients with AH and controls and correlated with clinical and pathological findings. RESULTS: Liver biopsies from patients with AH revealed neutrophils in contact with bile ducts, which correlated with biochemical and histological parameters of cholestasis. Cholangiocytes co-cultured with neutrophils lost ITPR3, and neutrophils from patients with AH were more potent than control neutrophils. Biochemical and histological findings were recapitulated in an AH animal model. Loss of ITPR3 was attenuated by neutrophils in which surface membrane proteins were removed. RNA-seq analysis implicated integrin ß1 (ITGB1) in neutrophil-cholangiocyte interactions and interference with ITGB1 on cholangiocytes blocked the ability of neutrophils to reduce cholangiocyte ITPR3 expression. Cell adhesion molecules on neutrophils interacted with ITGB1 to trigger RAC1-induced JNK activation, causing a c-Jun-mediated decrease in ITPR3 in cholangiocytes. CONCLUSIONS: Neutrophils bind to ITGB1 on cholangiocytes to contribute to cholestasis in AH. This previously unrecognised role for cholangiocytes in this disease alters our understanding of its pathogenesis and identifies new therapeutic targets.