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Fatty liver disease has grown into a major global health burden, attributed to multi-factors including sedentary lifestyle, obesogenic diet and prevalence of metabolic disorders. The lack of robust experimental models is hampering the research and therapeutic development for fatty liver disease. This study aims to develop an organoid-based 3D culture model to recapitulate key features of fatty liver disease focusing on intracellular lipid accumulation and metabolic dysregulation. We used human liver-derived intrahepatic cholangiocyte organoids and hepatocyte differentiated organoids. These organoids were exposed to lactate, pyruvate, and octanoic acid (LPO) for inducing lipid accumulation and mitochondrial impairment. Lipid accumulation resulted in alternations of gene transcription with major effects on metabolic pathways, including triglyceride and glucose level increase, which is consistent with metabolic changes in fatty liver disease patients. Interestingly, lipid accumulation affected mitochondria as shown by morphological transitions, alternations in expression of mitochondrial encoded genes, and reduction of ATP production. Meanwhile, we found treatment with obeticholic acid and metformin can alleviate fat accumulation in organoids. This study demonstrated that LPO exposure can induce lipid accumulation and associated metabolic dysregulation in human liver-derived organoids. This provides an innovative model for studying fatty liver disease and testing potential therapeutics. KEY MESSAGES: Lactate, pyruvate, and octanoic acid induce lipid accumulation in liver organoids. Organoids of human compared to mouse origin are more efficient in lipid accumulation. Lipid accumulation dysregulates metabolic pathway and impairs mitochondrial function. Demonstrating a proof-of-concept for testing medications in organoids., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Cancer-associated fibroblasts (CAFs) play a key role in the cancer process, but the research progress is hampered by the paucity of preclinical models that are essential for mechanistic dissection of cancer cell-CAF interactions. Here, we aimed to establish 3-dimensional (3D) organotypic co-cultures of primary liver tumor-derived organoids with CAFs, and to understand their interactions and the response to treatment., Methods: Liver tumor organoids and CAFs were cultured from murine and human primary liver tumors. 3D co-culture models of tumor organoids with CAFs and Transwell culture systems were established in vitro. A xenograft model was used to investigate the cell-cell interactions in vivo. Gene expression analysis of CAF markers in our hepatocellular carcinoma cohort and an online liver cancer database indicated the clinical relevance of CAFs., Results: To functionally investigate the interactions of liver cancer cells with CAFs, we successfully established murine and human 3D co-culture models of liver tumor organoids with CAFs. CAFs promoted tumor organoid growth in co-culture with direct cell-cell contact and in a Transwell system via paracrine signaling. Vice versa, cancer cells secrete paracrine factors regulating CAF physiology. Co-transplantation of CAFs with liver tumor organoids of mouse or human origin promoted tumor growth in xenograft models. Moreover, tumor organoids conferred resistance to clinically used anticancer drugs including sorafenib, regorafenib, and 5-fluorouracil in the presence of CAFs, or the conditioned medium of CAFs., Conclusions: We successfully established murine and human 3D co-culture models and have shown robust effects of CAFs in liver cancer nurturing and treatment resistance., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry−/−RAG2−/− mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids. [ABSTRACT FROM AUTHOR]

Background: The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. Methods: We stimulate a total of 106 colonic biopsies from 19 Crohn’s disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-κB activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1ß (IL-1ß) mRNA levels by qPCR and in cell culture models by NF-κB reporter-driven luciferase activity and Western blot for signal transduction elements. Results: We observe a strict inverse correlation between colonic epithelial PXR levels and NF-κB target gene expression in colonic biopsies from Crohn’s disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-κB activation in IBD. The correlation between colonic epithelial PXR levels and NF-κB target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-κB-dependent signal transduction whereas conversely NF-κB signaling reduces levels of PXR expression. Conclusions: Our data indicate that the PXR is a major and clinically relevant antagonist of NF-κB activity in the intestinal epithelial compartment during inflammatory bowel disease. [ABSTRACT FROM AUTHOR]