Canine Organoid Research Articles

Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach

Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.

Culture and characterization of canine and feline corneal epithelial organoids: A new tool for the study and treatment of corneal diseases

In this study, we isolated and cultured canine and feline 3D corneal organoids. Samples derived from corneal limbal epithelium from one canine and one feline patient were obtained by enucleation after euthanasia. Stem cell isolation and organoid culture were performed by culturing organoids in Matrigel. Organoids were subsequently embedded in paraffin for further characterization. The expression of key corneal epithelial and stromal cell markers in canine and feline organoids was evaluated at the mRNA level by RNA-ISH and at the protein level by immunofluorescence (IF) and immunohistochemistry (IHC), while histochemical analysis was performed on both tissues and organoids using periodic-acid Schiff (PAS), Sirius Red, Gomori's Trichrome, and Colloidal Iron stains. IF showed consistent expression of AQP1 within canine and feline organoids and tissues. P63 was present in canine tissues, canine organoids, and feline tissues, but not in feline organoids. Results from IHC staining further confirmed the primarily epithelial origin of the organoids. Canine and feline 3D corneal organoids can successfully be cultured and maintained and express epithelial and stem cell progenitor markers typical of the cornea. This novel in vitro model can be used in veterinary ophthalmology disease modeling, corneal drug testing, and regenerative medicine.

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells. The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol describes the tasks directly preceding an experiment, including in vitro validation of experimental results. Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability. Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells. The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocoldescribes the tasks directly preceding an experiment, including in vitro validation of experimental results. Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Standardization and Maintenance of 3D Canine Hepatic and Intestinal Organoid Cultures for Use in Biomedical Research.

Dogs develop complex multifactorial diseases analogous to humans, including inflammatory diseases, metabolic diseases, and cancer. Therefore, they represent relevant large animal models with the translational potential to human medicine. Organoids are 3-dimensional (3D), self-assembled structures derived from stem cells that mimic the microanatomy and physiology of their organ of origin. These translational in vitro models can be used for drug permeability and discovery applications, toxicology assessment, and to provide a mechanistic understanding of the pathophysiology of multifactorial chronic diseases. Furthermore, canine organoids can enhance the lives of companion dogs, providing input in various areas of veterinary research and facilitating personalized treatment applications in veterinary medicine. A small group of donors can create a biobank of organoid samples, reducing the need for continuous tissue harvesting, as organoid cell lines can be sub-cultured indefinitely. Herein, three protocols that focus on the culture of intestinal and hepatic canine organoids derived from adult stem cells are presented. The Canine Organoid Isolation Protocol outlines methods to process tissue and embedding of the cell isolate in a supportive matrix (solubilized extracellular membrane matrix). The Canine Organoid Maintenance Protocol describes organoid growth and maintenance, including cleaning and passaging along with appropriate timing for expansion. The Organoid Harvesting and Biobanking Protocol describes ways to extract, freeze, and preserve organoids for further analysis.

Harnessing The Biology of Intestinal Organoids To Accelerate Drug Discovery in Inflammatory Bowel Disease: A One Health Approach

Rationale and SignificanceCanine inflammatory bowel disease (IBD) refers to a group of chronic gastrointestinal (GI) disorders of unknown cause and pathogenesis which mimic the spectrum of chronic enteropathies in human patients with IBD. There is currently no cure for IBD and many therapeutic interventions developed over the past 3 decades have consistently failed to demonstrate evidence of efficacy in clinical trials. There is, therefore, a critical need to develop robust drug screening tools to accelerate the availability of effective and safe therapeutic strategies for management of IBD. With the aim of modeling spontaneous GI disease in dogs and humans, intestinal stem cell (ISC)‐derived organoids are emerging as a promising ex vivo tool in the study of IBD pathogenesis. Our working testable hypothesis is that canine organoids will faithfully reproduce structural and functional changes of the intestinal epithelium in dogs with IBD, which will enable more accurate prediction of therapeutic drug efficacy and safety. Long‐term, this model will provide critical information for the design of canine clinical trials, and ultimately generate preclinical data for similar studies in humans with IBD. Altogether, we therefore expect to contribute to decreased morbidity/mortality, and improved quality of life in dogs and patients with IBD.MethodsISCs isolated from endoscopic biopsies of two healthy dogs and two dogs with active IBD were differentiated into intestinal organoids. Ileal organoids and matching tissues were probed by RNA in situ hybridization and immunohistochemistry for phenotypic changes in IBD. A panel of six phenotypic markers identified different epithelial cell lineages (LGR5+: intestinal stem cell, ALP: enterocyte, PAS: goblet cell, NeuroG3: enteroendocrine cell), epithelial barrier integrity (ZO‐1) and cell proliferation (Ki‐67). Functional features of IBD organoids were investigated by cystic fibrosis transmembrane conductance regulator (CFTR) organoid swelling assay to measure Cl− channel‐water conductance.ResultsOur preliminary findings suggest that IBD organoids exhibit increased cell proliferation, intestinal stem cells, enteroendocrine cells, and expression of tight junction protein as compared to healthy organoids. Similar trends were observed when comparing phenotypic marker expression in original intestinal tissues. Forskolin significantly (P < 0.05) increased swelling of IBD organoids after 1 and 4 hr vs. controls indicating functional CFTR in organoids.ConclusionOverall, our data show that ileal organoids derived from dogs with IBD recapitulate both the phenotypic and physiological features of diseased tissue compared to healthy tissue, demonstrating its utility as an ex vivo model for investigating mechanism and therapeutic strategies in IBD. These preliminary results will be further validated by additional intestinal biopsies from 14 dogs with IBD in an ongoing clinical trials.Support or Funding Information Research Support: IG BiosciencesPreliminary phenotypic characterization of IBD organoids.(Left) Representative RNA ISH of ALP and NeuroG3 staining in ileal organoids at x40 magnification (ratio of total area). Scale bar: 50 μm. (Right) Summary box plots of ALP and PAS expression in IBD vs. Control (CTR) organoids. Data from 10 distinct microscopic fields were aggregated per dog.Figure 1

Abstract 3870: Validation of a canine intestinal organoid model as a preclinical screen to assess GI toxicity in novel oncology drug development

Abstract Gastrointestinal (GI) toxicity is a common and often severe dose-limiting side effect of chemotherapy. Symptoms include diarrhea, dehydration and ulceration leading to increased susceptibility to infection, due in part to the damage or loss of crypt and/or villi structures in the small intestine. As improvements in oncology therapeutics are pursued to acquire more efficacious agents, assessment of their potential GI toxicity is therefore crucial. The mouse small intestinal in vitro organoid model was first described by Sato et al. in 2009 and has been used to help understand the intestinal stem cell hierarchy and their regulatory genes. In parallel the pharmaceutical industry has been gaining interest, with the aim of validating organoids as a screening step prior to in vivo studies. We have previously developed and validated the organoid model as a screening tool to predict GI toxicity or mucosal regeneration in three species: mouse, rat and human. These techniques have recently been employed with canine tissue, successfully establishing canine organoids and assessing their suitability in our screening models in comparison to other species. The intestinal organoid culture conditions were designed to mimic the stem cell niche allowing cell differentiation and proliferation to occur. All expected intestinal lineages were present in the canine organoids and the epithelial hierarchy closely resembled that observed in vivo. The response of canine organoids to cytotoxic insult via treatment with common colorectal chemotherapy drugs correlated with their known responses in vivo. The organoid branches, which represent the crypts and contain the proliferative cells, were first lost upon treatment before complete organoid death, presumably due to the loss of stem cells within the branches. The level of toxicity associated with these chemotherapy agents was determined by calculation of IC50 values, which were then compared to published in vivo data, upon which correlation in drug sensitivity within the GI was observed. In summary, we conclude that the canine organoids, alongside other already validated organoid species, are a predictive preclinical model that can be used to identify any potential on-target, off-tissue GI toxicities induced by novel oncology therapeutics. Toxicity and mechanism of action can all be addressed in vitro to potentially reduce in vivo experimentation. Citation Format: Sarah M. Hoyle, Nicola Tonge, Charlotte Seaman, Mark Drinkwater, Adam Bonas, Vicky Murray-Tait, Aude-Marine Bonavita, Cath Booth. Validation of a canine intestinal organoid model as a preclinical screen to assess GI toxicity in novel oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3870.

Canine Models for Copper Homeostasis Disorders.

Copper is an essential trace nutrient metal involved in a multitude of cellular processes. Hereditary defects in copper metabolism result in disorders with a severe clinical course such as Wilson disease and Menkes disease. In Wilson disease, copper accumulation leads to liver cirrhosis and neurological impairments. A lack in genotype-phenotype correlation in Wilson disease points toward the influence of environmental factors or modifying genes. In a number of Non-Wilsonian forms of copper metabolism, the underlying genetic defects remain elusive. Several pure bred dog populations are affected with copper-associated hepatitis showing similarities to human copper metabolism disorders. Gene-mapping studies in these populations offer the opportunity to discover new genes involved in copper metabolism. Furthermore, due to the relatively large body size and long life-span of dogs they are excellent models for development of new treatment strategies. One example is the recent use of canine organoids for disease modeling and gene therapy of copper storage disease. This review addresses the opportunities offered by canine genetics for discovery of genes involved in copper metabolism disorders. Further, possibilities for the use of dogs in development of new treatment modalities for copper storage disorders, including gene repair in patient-derived hepatic organoids, are highlighted.