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1.
Nature ; 603(7902): 706-714, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35104837

RESUMO

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis.


Assuntos
COVID-19/patologia , COVID-19/virologia , Fusão de Membrana , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidade , Serina Endopeptidases/metabolismo , Internalização do Vírus , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Convalescença , Feminino , Humanos , Soros Imunes/imunologia , Intestinos/patologia , Intestinos/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Mutação , Mucosa Nasal/patologia , Mucosa Nasal/virologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Técnicas de Cultura de Tecidos , Virulência , Replicação Viral
2.
Gastroenterology ; 167(6): 1183-1197.e16, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38992449

RESUMO

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC), often associated with inflammatory bowel disease (IBD), presents a multifactorial etiology involving genetic, immunologic, and environmental factors. Gut dysbiosis and bacterial translocation have been implicated in PSC-IBD, yet the precise mechanisms underlying their pathogenesis remain elusive. Here, we describe the role of gut pathobionts in promoting liver inflammation and fibrosis due to the release of bacterial outer membrane vesicles (OMVs). METHODS: Preclinical mouse models in addition to ductal organoids were used to acquire mechanistic data. A proof-of-concept study including serum and liver biopsies of a patient cohort of PSC (n = 22), PSC-IBD (n = 45), and control individuals (n = 27) was performed to detect OMVs in the systemic circulation and liver. RESULTS: In both preclinical model systems and in patients with PSC-IBD, the translocation of OMVs to the liver correlated with enhanced bacterial sensing and accumulation of the NLRP3 inflammasome. Using ductal organoids, we were able to precisely attribute the pro-inflammatory and pro-fibrogenic properties of OMVs to signaling pathways dependent on Toll-like receptor 4 and NLRP3-gasdermin-D. The immunostimulatory potential of OMVs could be confirmed in macrophages and hepatic stellate cells. Furthermore, when we administered gut pathobiont-derived OMVs to Mdr2-/- mice, we observed a significant enhancement in liver inflammation and fibrosis. In a translational approach, we substantiated the presence of OMVs in the systemic circulation and hepatic regions of severe fibrosis using a PSC-IBD patient cohort. CONCLUSIONS: This study demonstrates the contribution of gut pathobionts in releasing OMVs that traverse the mucosal barrier and, thus, promote liver inflammation and fibrosis in PSC-IBD. OMVs might represent a critical new environmental factor that interacts with other disease factors to cause inflammation and thus define potential new targets for fibrosis therapy.


Assuntos
Colangite Esclerosante , Modelos Animais de Doenças , Microbioma Gastrointestinal , Doenças Inflamatórias Intestinais , Cirrose Hepática , Fígado , Colangite Esclerosante/imunologia , Colangite Esclerosante/microbiologia , Colangite Esclerosante/patologia , Animais , Humanos , Camundongos , Doenças Inflamatórias Intestinais/microbiologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Cirrose Hepática/patologia , Cirrose Hepática/microbiologia , Cirrose Hepática/imunologia , Cirrose Hepática/metabolismo , Cirrose Hepática/etiologia , Fígado/patologia , Fígado/imunologia , Translocação Bacteriana , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Disbiose , Membrana Externa Bacteriana/metabolismo , Organoides , Estudos de Casos e Controles , Estudo de Prova de Conceito , Camundongos Endogâmicos C57BL , Feminino , Transdução de Sinais , Masculino , Camundongos Knockout
3.
Hepatology ; 77(1): 305-322, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-35596930

RESUMO

The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions.


Assuntos
Gastroenterologia , Humanos , Organoides , Fígado , Medicina Regenerativa , Hepatócitos
4.
Dig Dis Sci ; 69(10): 3810-3823, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39160386

RESUMO

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls. METHODS: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature. RESULTS: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1. CONCLUSIONS: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.


Assuntos
Colangite Esclerosante , Perfilação da Expressão Gênica , Organoides , Humanos , Colangite Esclerosante/genética , Colangite Esclerosante/patologia , Organoides/metabolismo , Organoides/patologia , Perfilação da Expressão Gênica/métodos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Ductos Biliares/patologia , Análise de Célula Única/métodos , Transcriptoma , Masculino , Feminino
5.
J Intern Med ; 293(5): 636-647, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37018129

RESUMO

BACKGROUND AND AIMS: Studies have demonstrated that reducing farnesoid X receptor activity with ursodeoxycholic acid (UDCA) downregulates angiotensin-converting enzyme in human lung, intestinal and cholangiocytes organoids in vitro, in human lungs and livers perfused ex situ, reducing internalization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell. This offers a potential novel target against coronavirus disease 2019 (COVID-19). The objective of our study was to compare the association between UDCA exposure and SARS-CoV-2 infection, as well as varying severities of COVID-19, in a large national cohort of participants with cirrhosis. METHODS: In this retrospective cohort study among participants with cirrhosis in the Veterans Outcomes and Costs Associated with Liver cohort, we compared participants with exposure to UDCA, with a propensity score (PS) matched group of participants without UDCA exposure, matched for clinical characteristics, and vaccination status. The outcomes included SARS-CoV-2 infection, symptomatic, at least moderate, severe, or critical COVID-19, and COVID-19-related death. RESULTS: We compared 1607 participants with cirrhosis who were on UDCA, with 1607 PS-matched controls. On multivariable logistic regression, UDCA exposure was associated with reduced odds of developing SARS-CoV-2 infection (adjusted odds ratio [aOR] 0.54, 95% confidence interval [CI] 0.41-0.71, p < 0.0001). Among patients who developed COVID-19, UDCA use was associated with reduced disease severity, including symptomatic COVID-19 (aOR 0.54, 95% CI 0.39-0.73, p < 0.0001), at least moderate COVID-19 (aOR 0.51, 95% CI 0.32-0.81, p = 0.005), and severe or critical COVID-19 (aOR 0.48, 95% CI 0.25-0.94, p = 0.03). CONCLUSIONS: In participants with cirrhosis, UDCA exposure was associated with both a decrease in SARS-CoV-2 infection, and reduction in symptomatic, at least moderate, and severe/critical COVID-19.


Assuntos
COVID-19 , Cirrose Hepática Biliar , Humanos , Ácido Ursodesoxicólico/uso terapêutico , COVID-19/complicações , Estudos Retrospectivos , SARS-CoV-2 , Cirrose Hepática/complicações , Cirrose Hepática/tratamento farmacológico
6.
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
7.
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
9.
Development ; 143(23): 4405-4418, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27899508

RESUMO

Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Ciclina D/genética , Proteínas Fetais/genética , Engenharia Genética/métodos , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Proteínas com Domínio T/genética , Diferenciação Celular/genética , Células Cultivadas , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fatores de Transcrição
10.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt B): 1532-1538, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29097260

RESUMO

The generation of bioengineered biliary tissue could contribute to the management of some of the most impactful cholangiopathies associated with liver transplantation, such as biliary atresia or ischemic cholangiopathy. Recent advances in tissue engineering and in vitro cholangiocyte culture have made the achievement of this goal possible. Here we provide an overview of these developments and review the progress towards the generation and transplantation of bioengineered bile ducts. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni and Peter Jansen.


Assuntos
Doenças dos Ductos Biliares/cirurgia , Ductos Biliares/transplante , Órgãos Bioartificiais , Engenharia Tecidual/métodos , Animais , Ductos Biliares/citologia , Engenharia Biomédica/métodos , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Modelos Animais de Doenças , Células Epiteliais , Humanos , Transplante de Fígado/efeitos adversos , Transplante de Fígado/métodos , Técnicas de Cultura de Órgãos/métodos
11.
Hepatology ; 62(1): 303-11, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25502113

RESUMO

Liver disease is a leading cause of death in the Western world. However, our insight into the underlying disease mechanisms and the development of novel therapeutic agents has been hindered by limited availability of primary tissue, intraspecies variability associated with the use of animal models, and reduced long-term viability of isolated and diseased liver cells. The emergence of human induced pluripotent stem cells and differentiation protocols to generate hepatocyte-like cells has opened the possibility of addressing these issues. Here, we discuss the recent progress and potential in the production of various cell types constituting the liver and their applications to model liver diseases and test drug toxicity in vitro.


Assuntos
Hepatócitos , Células-Tronco Pluripotentes Induzidas , Hepatopatias , Doença Hepática Induzida por Substâncias e Drogas , Humanos , Deficiências na Proteostase
12.
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
13.
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
14.
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
15.
Nat Med ; 26(5): 681-687, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32327758

RESUMO

We investigated SARS-CoV-2 potential tropism by surveying expression of viral entry-associated genes in single-cell RNA-sequencing data from multiple tissues from healthy human donors. We co-detected these transcripts in specific respiratory, corneal and intestinal epithelial cells, potentially explaining the high efficiency of SARS-CoV-2 transmission. These genes are co-expressed in nasal epithelial cells with genes involved in innate immunity, highlighting the cells' potential role in initial viral infection, spread and clearance. The study offers a useful resource for further lines of inquiry with valuable clinical samples from COVID-19 patients and we provide our data in a comprehensive, open and user-friendly fashion at www.covid19cellatlas.org.

17.
Dig Liver Dis ; 51(6): 753-760, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30948332

RESUMO

Most of the research behind new medical advances is carried out using either animal models or cancer cells, which both have their disadvantage in particular with regard to medical applications such as personalized medicine and novel therapeutic approaches. However, recent advances in stem cell biology have enabled long-term culturing of organotypic intestinal or hepatic tissues derived from tissue resident or pluripotent stem cells. These 3D structures, denoted as organoids, represent a substantial advance in structural and functional complexity over traditional in vitro cell culture models that are often non-physiological and transformed. They can recapitulate the in vivo architecture, functionality and genetic signature of the corresponding tissue. The opportunity to model epithelial cell biology, epithelial turnover, barrier dynamics, immune-epithelial communication and host-microbe interaction more efficiently than previous culture systems, greatly enhance the translational potential of organotypic hepato-gastrointestinal culture systems. Thus there is increasing interest in using such cultured cells as a source for tissue engineering, regenerative medicine and personalized medicine. This review will highlight some of the established and also some exciting novel perspectives on organoids in the fields of gastroenterology and hepatology.


Assuntos
Doença , Células-Tronco Pluripotentes Induzidas/citologia , Modelos Biológicos , Organoides/citologia , Organoides/fisiologia , Animais , Gastroenterologia/tendências , Humanos , Especificidade de Órgãos , Medicina de Precisão/métodos , Medicina Regenerativa/métodos , Pesquisa Translacional Biomédica
18.
Methods Mol Biol ; 1981: 373-382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016668

RESUMO

Cholangiocytes play a crucial role in the pathophysiology of cholestasis. However, research on human cholangiocytes has been restricted by challenges in long-term propagation and large-scale expansion of primary biliary epithelium. The advent of organoid technology has overcome this limitation allowing long-term culture of a variety of epithelia from multiple organs. Here, we describe two methods for growing human cholangiocytes in organoid format. The first applies to the generation of intrahepatic bile ducts using human induced pluripotent stem cells using a protocol of differentiation that recapitulates physiological bile duct development. The second method allows the propagation of primary biliary epithelium from the extrahepatic ducts or gallbladder. Both protocols result in large numbers of cholangiocyte organoids expressing biliary markers and maintaining key cholangiocyte functions.


Assuntos
Colestase/metabolismo , Colestase/patologia , Organoides/patologia , Ductos Biliares/metabolismo , Ductos Biliares/patologia , Vesícula Biliar/metabolismo , Vesícula Biliar/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fígado/metabolismo , Fígado/patologia , Organoides/metabolismo
19.
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
20.
Nat Biotechnol ; 37(8): 895-906, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31375810

RESUMO

The epicardium and its derivatives provide trophic and structural support for the developing and adult heart. Here we tested the ability of human embryonic stem cell (hESC)-derived epicardium to augment the structure and function of engineered heart tissue in vitro and to improve efficacy of hESC-cardiomyocyte grafts in infarcted athymic rat hearts. Epicardial cells markedly enhanced the contractility, myofibril structure and calcium handling of human engineered heart tissues, while reducing passive stiffness compared with mesenchymal stromal cells. Transplanted epicardial cells formed persistent fibroblast grafts in infarcted hearts. Cotransplantation of hESC-derived epicardial cells and cardiomyocytes doubled graft cardiomyocyte proliferation rates in vivo, resulting in 2.6-fold greater cardiac graft size and simultaneously augmenting graft and host vascularization. Notably, cotransplantation improved systolic function compared with hearts receiving either cardiomyocytes alone, epicardial cells alone or vehicle. The ability of epicardial cells to enhance cardiac graft size and function makes them a promising adjuvant therapeutic for cardiac repair.


Assuntos
Coração/fisiologia , Células-Tronco Embrionárias Humanas , Infarto do Miocárdio/terapia , Miócitos Cardíacos , Regeneração , Animais , Embrião de Galinha , Regulação da Expressão Gênica , Humanos , Masculino , Ratos , Ratos Nus , Ratos Sprague-Dawley , Engenharia Tecidual
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