SummaryThe recent development of 3D-liver stem cell cultures (hepatic organoids) opens up new avenues for gene and/or stem cell therapy to treat liver disease. To test safety and efficacy, a relevant large animal model is essential but not yet established. Because of its shared pathologies and disease pathways, the dog is considered the best model for human liver disease. Here we report the establishment of a long-term canine hepatic organoid culture allowing undifferentiated expansion of progenitor cells that can be differentiated toward functional hepatocytes. We show that cultures can be initiated from fresh and frozen liver tissues using Tru-Cut or fine-needle biopsies. The use of Wnt agonists proved important for canine organoid proliferation and inhibition of differentiation. Finally, we demonstrate that successful gene supplementation in hepatic organoids of COMMD1-deficient dogs restores function and can be an effective means to cure copper storage disease.

SummaryHepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research.

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The shortage of liver organ donors is increasing and the need for viable alternatives is urgent. Liver cell (hepatocyte) transplantation may be a less invasive treatment compared with liver transplantation. Unfortunately, hepatocytes cannot be expanded in vitro, and allogenic cell transplantation requires long-term immunosuppression. Organoid-derived adult liver stem cells can be cultured indefinitely to create sufficient cell numbers for transplantation, and they are amenable to gene correction. This study provides preclinical proof of concept of the potential of cell transplantation in a large animal model of inherited copper toxicosis, such as Wilson’s disease, a Mendelian disorder that causes toxic copper accumulation in the liver. Hepatic progenitors from five COMMD1-deficient dogs were isolated and cultured using the 3D organoid culture system. After genetic restoration of COMMD1 expression, the organoid-derived hepatocyte-like cells were safely delivered as repeated autologous transplantations via the portal vein. Although engraftment and repopulation percentages were low, the cells survived in the liver for up to two years post-transplantation. The low engraftment was in line with a lack of functional recovery regarding copper excretion. This preclinical study confirms the survival of genetically corrected autologous organoid-derived hepatocyte-like cells in vivo and warrants further optimization of organoid engraftment and functional recovery in a large animal model of human liver disease.

Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.

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New curative therapies for severe liver disease are urgently needed in both the human and veterinary clinic. It is important to find new treatment modalities which aim to compensate for the loss of parenchymal tissue and to repopulate the liver with healthy hepatocytes. A prime focus in regenerative medicine of the liver is the use of adult liver stem cells, or hepatic progenitor cells (HPCs), for functional recovery of liver disease. This review describes recent developments in HPC research in dog and cat and compares these findings to experimental rodent studies and human pathology. Specifically, the role of HPCs in liver regeneration, key components of the HPC niche, and HPC activation in specific types of canine and feline liver disease will be reviewed. Finally, the potential applications of HPCs in regenerative medicine of the liver are discussed and a potential role is suggested for dogs as first target species for HPC-based trials.

Background: Hepatic lipidosis is increasing in incidence in the Western world, with cats being particularly sensitive. When cats stop eating and start utilizing their fat reserves, free fatty acids (FFAs) increase in blood, causing an accumulation of triacylglycerol (TAG) in the liver.Objective: Identifying potential new drugs that can be used to treat hepatic lipidosis in cats using a feline hepatic organoid system. Animals: Liver organoids obtained from 6 cats.Methods: Eight different drugs were tested, and the 2 most promising were further studied using a quantitative TAG assay, lipid droplet staining, and qPCR. Results: Both T863 (a diacylglycerol O-acyltransferase 1 [DGAT1] inhibitor) and 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR; an adenosine monophosphate kinase activator) decreased TAG accumulation by 55% (P < .0001) and 46% (P = .0003), respectively. Gene expression of perilipin 2 (PLIN2) increased upon the addition of FFAs to the medium and decreased upon treatment with AICAR but not significantly after treatment with T863.Conclusions and Clinical Importance: Two potential drugs useful in the treatment of hepatic lipidosis in cats were identified. The drug T863 inhibits DGAT1, indicating that DGAT1 is the primary enzyme responsible for TAG synthesis from external fatty acids in cat organoids. The drug AICAR may act as a lipid-lowering compound via decreasing PLIN2 mRNA. Liver organoids can be used as an in vitro tool for drug testing in a species-specific system and provide the basis for further clinical testing of drugs to treat steatosis. K E Y W O R D SAICAR, diacylglycerol O-acyltransferase 1 inhibitor, hepatic organoids, perilipin 2, steatosis Abbreviations: AICAR, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside; AMPK, adenosine monophosphate-activated protein kinase; AMPKK, AMPK kinase; CYP3A132, cytochrome 3A132; DAG, diacylglycerol; DGAT, diacylglycerol O-acyltransferase; FFA, free fatty acid; HMBS, hydroxymethylbilane synthase; HPRT-1, hypoxanthine phosphoribosyltransferase 1; LGR5, leucine-rich repeat-containing G protein-coupled receptor 5; NAFLD, non-alcoholic fatty liver disease; PLIN2, perilipin 2; RPS5, ribosomal protein S5; TAG, triacylglycerol; TTR, transthyretin; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta; ZMP, 5-amino-4-imidazolecarboxamide ribose-monophosphate.Maya W. Haaker and Hedwig S. Kruitwagen contributed equally to this work.