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1.
Nature ; 615(7950): 134-142, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36470304

RESUMO

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Receptores Virais , Ácido Ursodesoxicólico , Animais , Humanos , Camundongos , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/prevenção & controle , Receptores Virais/genética , Receptores Virais/metabolismo , Estudos Retrospectivos , SARS-CoV-2/metabolismo , Tratamento Farmacológico da COVID-19 , Cricetinae , Transcrição Gênica , Ácido Ursodesoxicólico/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Sistema de Registros , Reprodutibilidade dos Testes , Transplante de Fígado
2.
Hepatology ; 73(1): 247-267, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32222998

RESUMO

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


Assuntos
Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Intra-Hepáticos/citologia , Células Epiteliais/citologia , Organoides/fisiologia , Animais , Bile , Ductos Biliares Extra-Hepáticos/fisiologia , Ductos Biliares Intra-Hepáticos/fisiologia , Diferenciação Celular , Ducto Colédoco/citologia , Células Epiteliais/fisiologia , Vesícula Biliar/citologia , Regulação da Expressão Gênica , Humanos , Queratina-19/análise , Fígado/fisiologia , Camundongos , RNA-Seq , Obtenção de Tecidos e Órgãos
3.
J Hepatol ; 73(4): 918-932, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32535061

RESUMO

Our insight into the pathogenesis of cholestatic liver disease remains limited, partly owing to challenges in capturing the multitude of factors that contribute to disease pathogenesis in vitro. Tissue engineering could address this challenge by combining cells, materials and fabrication strategies into dynamic modelling platforms, recapitulating the multifaceted aetiology of cholangiopathies. Herein, we review the advantages and limitations of platforms for bioengineering the biliary tree, looking at how these can be applied to model biliary disorders, as well as exploring future directions for the field.


Assuntos
Sistema Biliar/patologia , Colestase/terapia , Engenharia Tecidual/métodos , Animais , Colestase/patologia , Células Epiteliais/patologia , Humanos
4.
Hepatol Commun ; 8(3)2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38407207

RESUMO

BACKGROUND: Ductular reactivity is central to the pathophysiology of cholangiopathies. Mechanisms underlying the reactive phenotype activation by exogenous inflammatory mediators and bile acids are poorly understood. METHODS: Using human extrahepatic cholangiocyte organoids (ECOs) we developed an injury model emulating the cholestatic microenvironment with exposure to inflammatory mediators and various pathogenic bile acids. Moreover, we explored roles for the bile acid activated Sphingosine-1-phosphate receptor 2 (S1PR2) and potential beneficial effects of therapeutic bile acids UDCA and norUDCA. RESULTS: Synergistic exposure to bile acids (taurocholic acid, glycocholic acid, glycochenodeoxycholic acid) and TNF-α for 24 hours induced a reactive state as measured by ECO diameter, proliferation, lactate dehydrogenase activity and reactive phenotype markers. While NorUDCA and UDCA treatments given 8 hours after injury induction both suppressed reactive phenotype activation and most injury parameters, proliferation was improved by NorUDCA only. Extrahepatic cholangiocyte organoid stimulation with S1PR2 agonist sphingosine-1-phosphate reproduced the cholangiocyte reactive state and upregulated S1PR2 downstream mediators; these effects were suppressed by S1PR2 antagonist JET-013 (JET), downstream mediator extracellular signal-regulated kinase 1/2 inhibitor, and by norUDCA or UDCA treatments. JET also partially suppressed reactive phenotype after bile acid injury. CONCLUSIONS: We developed a novel model to study the reactive cholangiocyte state in response to pathological stimuli in cholestasis and demonstrated a contributory role of S1PR2 signaling in both injury and NorUDCA/UDCA treatments. This model is a valuable tool to further explore the pathophysiology of human cholangiopathies.


Assuntos
Ácidos e Sais Biliares , Colestase , Humanos , Mediadores da Inflamação , Fenótipo , Transdução de Sinais
5.
Nat Cell Biol ; 24(10): 1487-1498, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36109670

RESUMO

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances have been hampered by the lack of knowledge concerning human hepatic development. Here, we addressed this limitation by describing the developmental trajectories of different cell types that make up the human liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing essential roles during organogenesis. We utilized this information to derive bipotential hepatoblast organoids and then exploited this model system to validate the importance of signalling pathways in hepatocyte and cholangiocyte specification. Further insights into hepatic maturation also enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a platform to investigate the basic mechanisms directing human liver development and to produce cell types for clinical applications.


Assuntos
Hepatócitos , Fígado , Humanos , Fígado/metabolismo , Hepatócitos/metabolismo , Diferenciação Celular , Organoides , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
6.
Science ; 371(6531): 839-846, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33602855

RESUMO

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.


Assuntos
Doenças dos Ductos Biliares/terapia , Ductos Biliares Intra-Hepáticos/fisiologia , Ductos Biliares/citologia , Terapia Baseada em Transplante de Células e Tecidos , Células Epiteliais/citologia , Organoides/transplante , Animais , Bile , Ductos Biliares/fisiologia , Ductos Biliares Intra-Hepáticos/citologia , Ducto Colédoco/citologia , Células Epiteliais/fisiologia , Vesícula Biliar/citologia , Regulação da Expressão Gênica , Humanos , Fígado/fisiologia , Transplante de Fígado , Transplante de Células-Tronco Mesenquimais , Camundongos , Organoides/fisiologia , RNA-Seq , Obtenção de Tecidos e Órgãos , Transcriptoma
7.
Nat Protoc ; 14(6): 1884-1925, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110298

RESUMO

Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies.


Assuntos
Ductos Biliares/citologia , Ductos Biliares/fisiologia , Organoides/citologia , Organoides/fisiologia , Regeneração , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Separação Celular/métodos , Células Cultivadas , Desenho de Equipamento , Humanos , Camundongos , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química
8.
Nat Med ; 23(8): 954-963, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28671689

RESUMO

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


Assuntos
Ductos Biliares Extra-Hepáticos/fisiologia , Células Epiteliais/citologia , Vesícula Biliar/fisiologia , Organoides/fisiologia , Regeneração/fisiologia , Engenharia Tecidual/métodos , Animais , Ductos Biliares Extra-Hepáticos/citologia , Ductos Biliares Extra-Hepáticos/lesões , Sistema Biliar/citologia , Sistema Biliar/lesões , Sistema Biliar/fisiologia , Transplante de Células , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Vesícula Biliar/lesões , Humanos , Técnicas In Vitro , Queratina-19/metabolismo , Queratina-7/metabolismo , Camundongos , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Secretina/farmacologia , Somatostatina/farmacologia , Alicerces Teciduais , gama-Glutamiltransferase/metabolismo
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