Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

Verstegen, Monique M A; Roos, Floris J.M.; Burka, Ksenia; Gehart, Helmuth; Jager, Myrthe; Wolf, Maaike de; Bijvelds, Marcel J. C.; Jonge, Hugo R. de; Ardisasmita, Arif Ibrahim; Huizen, Nick A. van; Roest, Henk P.; Jonge, Jeroen de; Koch, Michael; Pampaloni, Francesco; Fuchs, Sabine A.; Schene, Imre F.; Luider, Theo M.; Doef, Hubert P.J. van der; Bodewes, Frank; Kleine, Ruben H J de; Spee, Bart; Kremers, Gert‐Jan; Clevers, Hans; IJzermans, Jan N. M.; Cuppen, Edwin; Laan, Luc J. W. van der

doi:10.1038/s41598-020-79082-8

Cited by 50 publications

(77 citation statements)

References 42 publications

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“…The hICOs (n = 7), ALD-ICOs (n = 3), and PSC-ICOs (n = 3) were initiated and cultured as previously described. 16 , 17 Briefly, liver tissue biopsy specimens (0.5–1 cm 3 ) derived from donors’ or patients’ livers were minced, rinsed twice with Earle's balanced salt solution (Thermo-Fisher, Waltham, MA), and digested in collagenase solution (2.5 mg/mL collagenase Type A; Sigma-Aldrich, St. Louis, MO) in advanced Dulbecco’s modified Eagle medium (DMEM)/F-12 medium (Invitrogen, Carlsbad, CA) for 15 minutes at 37°C. The digestion was stopped by the addition of cold advanced DMEM/F-12, and then the single-cell suspension was filtered through a 70-μm nylon cell strainer and spun at 1500 rpm for 5 minutes at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

Shi

Verstegen

Roest

et al. 2022

Cellular and Molecular Gastroenterology and Hepatology

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Background & Aims Liver and bile duct diseases often are associated with extensive cell death of cholangiocytes. Necroptosis represents a common mode of programmed cell death in cholangiopathy, however, detailed mechanistic knowledge is limited owing to the lack of appropriate in vitro models. To address this void, we investigated whether human intrahepatic cholangiocyte organoids (ICOs) can recapitulate cholangiopathy-associated necroptosis and whether this model can be used for drug screening. Methods We evaluated the clinical relevance of necroptosis in end-stage liver diseases and liver transplantation by immunohistochemistry. Cholangiopathy-associated programmed cell death was evoked in ICOs derived from healthy donors or patients with primary sclerosing cholangitis or alcoholic liver diseases by the various stimuli. Results The expression of key necroptosis mediators, receptor-interacting protein 3 and phosphorylated mixed lineage kinase domain-like, in cholangiocytes during end-stage liver diseases was confirmed. The phosphorylated mixed lineage kinase domain-like expression was etiology-dependent. Gene expression analysis confirmed that primary cholangiocytes are more prone to necroptosis compared with primary hepatocytes. Both apoptosis and necroptosis could be specifically evoked using tumor necrosis factor α and second mitochondrial-derived activator of caspases mimetic, with or without caspase inhibition in healthy and patient-derived ICOs. Necroptosis also was induced by ethanol metabolites or human bile in ICOs from donors and patients. The organoid cultures further uncovered interdonor variable and species-specific drug responses. Dabrafenib was identified as a potent necroptosis inhibitor and showed a protective effect against ethanol metabolite toxicity. Conclusions Human ICOs recapitulate cholangiopathy-associated necroptosis and represent a useful in vitro platform for the study of biliary cytotoxicity and preclinical drug evaluation.

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Section: Methodsmentioning

confidence: 99%

“… 14 We and others previously described methods to culture and expand human and mouse cholangiocyte organoids, which are capable of self-organization and retain most biliary characteristics in vitro. 15 , 16 , 17 …”

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confidence: 99%

Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

Shi

Verstegen

Roest

et al. 2022

Cellular and Molecular Gastroenterology and Hepatology

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“…These organoids were shown to have a defective secretory response to forskolin because of loss of CFTR‐mediated chloride‐ion transport. ( 54 ) This model system could be useful to characterize the molecular defects observed with different CFTR mutations.…”

Section: Hepatobiliary Organoid Models and Their Applicationmentioning

confidence: 99%

Hepatobiliary Organoids and Their Applications for Studies of Liver Health and Disease: Are We There Yet?

2021

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Organoid culture systems have emerged as a frontier technology in liver and biliary research. These three‐dimensional (3D) cell cultures derived from pluripotent and adult hepatobiliary cells model organ structure and function. Building on gastrointestinal organoid establishment, hepatobiliary organoid cultures were generated from mouse leucine‐rich repeat–containing G‐protein–coupled receptor 5–positive liver progenitor cells. Subsequently, 3D hepatobiliary organoid cultures were developed from hepatocytes and cholangiocytes to model human and animal hepatobiliary health and disease. Hepatocyte organoids have been used to study Alagille syndrome, fatty liver disease, Wilson disease, hepatitis B viral infection, and cystic fibrosis. Cholangiocyte organoids have been established to study normal cholangiocyte biology and primary sclerosing cholangitis and to test organoid potential to form bile ducts and gallbladder tissue in vitro. Hepatobiliary cancer organoids, termed tumoroids, have been established from frozen and fresh human tissues and used as a drug‐testing platform and for biobanking of cancer samples. CRISPR‐based gene modifications and organoid exposure to infectious agents have permitted the generation of organoid models of carcinogenesis. This review summarizes currently available adult cell–derived hepatobiliary organoid models and their applications. Challenges faced by this young technology will be discussed, including the cellular immaturity of organoid‐derived hepatocytes, co‐culture development to better model complex tissue structure, the imperfection of extracellular matrices, and the absence of standardized protocols and model validation.

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“…This organ-on-chip system will thus allow broadening the concept of personalized medicine for CF from the well-established primary lung and intestinal cultures to a pancreatic model for studying epithelial and/or endocrine dysfunctions. Even more organ-specific cell models are emerging, such as extrahepatic and intrahepatic cholangiocyte organoids ( Verstegen et al, 2020 ), which collectively will allow to better understand the pathological mechanisms and study a therapeutic rescue in the many organs affected in CF.…”

Section: Cell and Animal Models Of Diseasementioning

confidence: 99%

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis

et al. 2021

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Cystic fibrosis (CF) is a severe genetic disease for which curative treatment is still lacking. Next generation biotechnologies and more efficient cell-based and in vivo disease models are accelerating the development of novel therapies for CF. Gene editing tools, like CRISPR-based systems, can be used to make targeted modifications in the genome, allowing to correct mutations directly in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Alternatively, with these tools more relevant disease models can be generated, which in turn will be invaluable to evaluate novel gene editing-based therapies for CF. This critical review offers a comprehensive description of currently available tools for genome editing, and the cell and animal models which are available to evaluate them. Next, we will give an extensive overview of proof-of-concept applications of gene editing in the field of CF. Finally, we will touch upon the challenges that need to be addressed before these proof-of-concept studies can be translated towards a therapy for people with CF.

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Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

Cited by 50 publications

References 42 publications

Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

Recapitulating Cholangiopathy-Associated Necroptotic Cell Death In Vitro Using Human Cholangiocyte Organoids

Hepatobiliary Organoids and Their Applications for Studies of Liver Health and Disease: Are We There Yet?

On the Corner of Models and Cure: Gene Editing in Cystic Fibrosis

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