Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Derivation of adult canine intestinal organoids for translational research in gastroenterology.

BACKGROUND: Large animal models, such as the dog, are increasingly being used for studying diseases including gastrointestinal (GI) disorders. Dogs share similar environmental, genomic, anatomical, and intestinal physiologic features with humans. To bridge the gap between commonly used animal models, such as rodents, and humans, and expand the translational potential of the dog model, we developed a three-dimensional (3D) canine GI organoid (enteroid and colonoid) system. Organoids have recently gained interest in translational research as this model system better recapitulates the physiological and molecular features of the tissue environment in comparison with two-dimensional cultures. RESULTS: Organoids were derived from tissue of more than 40 healthy dogs and dogs with GI conditions, including inflammatory bowel disease (IBD) and intestinal carcinomas. Adult intestinal stem cells (ISC) were isolated from whole jejunal tissue as well as endoscopically obtained duodenal, ileal, and colonic biopsy samples using an optimized culture protocol. Intestinal organoids were comprehensively characterized using histology, immunohistochemistry, RNA in situ hybridization, and transmission electron microscopy, to determine the extent to which they recapitulated the in vivo tissue characteristics. Physiological relevance of the enteroid system was defined using functional assays such as optical metabolic imaging (OMI), the cystic fibrosis transmembrane conductance regulator (CFTR) function assay, and Exosome-Like Vesicles (EV) uptake assay, as a basis for wider applications of this technology in basic, preclinical and translational GI research. We have furthermore created a collection of cryopreserved organoids to facilitate future research. CONCLUSIONS: We establish the canine GI organoid systems as a model to study naturally occurring intestinal diseases in dogs and humans, and that can be used for toxicology studies, for analysis of host-pathogen interactions, and for other translational applications.

Effect of amlodipine on the circulating renin-angiotensin-aldosterone system in healthy cats.

BACKGROUND: Systemic hypertension (SH) is a common cardiovascular disease in older cats that is treated primarily with the calcium channel blocker amlodipine besylate (AML). The systemic effect of AML on the classical and alterative arms of the renin-angiotensin-aldosterone system (RAAS) in cats is incompletely characterized. HYPOTHESIS/OBJECTIVES: To determine the effect of AML compared to placebo on circulating RAAS biomarkers in healthy cats using RAAS fingerprinting. ANIMALS: Twenty healthy client-owned cats. METHODS: Cats were administered amlodipine besylate (0.625 mg in toto) or placebo by mouth once daily for 14 days in a crossover design with a 4-week washout period. Plasma AML concentrations and RAAS biomarker concentrations were measured at multiple timepoints after the final dose in each treatment period. Time-weighted averages for RAAS biomarkers over 24 hours after dosing were compared between treatment groups using Wilcoxon rank-sum testing. RESULTS: Compared to placebo, AML treatment was associated with increases in markers of plasma renin concentration (median 44% increase; interquartile range [IQR] 19%-86%; P = .009), angiotensin I (59% increase; IQR 27-101%; P = .006), angiotensin II (56% increase; IQR 5-70%; P = .023), angiotensin IV (42% increase; -19% to 89%; P = .013); and angiotensin 1-7 (38% increase; IQR 9-118%; P = .015). CONCLUSIONS AND CLINICAL IMPORTANCE: In healthy cats, administration of AML resulted in nonspecific activation of both classical and alternative RAAS pathways.

Preclinical modeling of metabolic syndrome to study the pleiotropic effects of novel antidiabetic therapy independent of obesity.

Cardiovascular-kidney-metabolic health reflects the interactions between metabolic risk factors, chronic kidney disease, and the cardiovascular system. A growing body of literature suggests that metabolic syndrome (MetS) in individuals of normal weight is associated with a high prevalence of cardiovascular diseases and an increased mortality. The aim of this study was to establish a non-invasive preclinical model of MetS in support of future research focusing on the effects of novel antidiabetic therapies beyond glucose reduction, independent of obesity. Eighteen healthy adult Beagle dogs were fed an isocaloric Western diet (WD) for ten weeks. Biospecimens were collected at baseline (BAS1) and after ten weeks of WD feeding (BAS2) for measurement of blood pressure (BP), serum chemistry, lipoprotein profiling, blood glucose, glucagon, insulin secretion, NT-proBNP, angiotensins, oxidative stress biomarkers, serum, urine, and fecal metabolomics. Differences between BAS1 and BAS2 were analyzed using non-parametric Wilcoxon signed-rank testing. The isocaloric WD model induced significant variations in several markers of MetS, including elevated BP, increased glucose concentrations, and reduced HDL-cholesterol. It also caused an increase in circulating NT-proBNP levels, a decrease in serum bicarbonate, and significant changes in general metabolism, lipids, and biogenic amines. Short-term, isocaloric feeding with a WD in dogs replicated key biological features of MetS while also causing low-grade metabolic acidosis and elevating natriuretic peptides. These findings support the use of the WD canine model for studying the metabolic effects of new antidiabetic therapies independent of obesity.

Changes in symmetric dimethylarginine and glomerular filtration rates in hyperthyroid cats undergoing radioiodine therapy.

OBJECTIVES: The aims of this study were to evaluate concentrations of symmetric dimethylarginine (SDMA) in hyperthyroid cats before and after radioiodine treatment, and to compare results with other variables used to assess kidney function in cats (creatinine, urine specific gravity [USG] and glomerular filtration rate [GFR] measured by renal scintigraphy). METHODS: Thirteen cats diagnosed with hyperthyroidism based on clinical signs and increased serum total thyroxine (TT4) were included in this prospective study. Study design included physical examination, complete blood count, serum chemistry, TT4, urinalysis and SDMA before treatment (T0) and at 1 month (T1) and 3 months post-treatment (T3). GFR was quantified by renal scintigraphy at T0 and T3. RESULTS: Median GFR decreased significantly from baseline (3.18 ml/kg/min; range 1.35-4.87) at T3 (2.22 ml/kg/min; range 1.81-3.42 [P = 0.005]). While median creatinine and serum urea nitrogen increased post-treatment (creatinine: T0 = 0.8 mg/dl [range 0.4-1.1], T1 = 1.3 mg/dl [range 0.9-2]; T3 = 1.65 mg/dl [range 0.8-2.8]; P <0.001; serum urea nitrogen: T0 = 23 mg/dl [range 15-26]; T1 = 27 mg/dl [range 20-40]; T3 = 27.5 mg/dl [range 20-36]; P <0.001), SDMA and USG did not change significantly (SDMA: T0 = 11 µg/dl [range 7-15]; T1 = 12 µg/dl [range 6-16]; T3 = 10.5 µg/dl [range 8-21]; P = 0.789; USG: T0 = 1.030 [range 1.011-1.059]; T1 = 1.035 [range 1.012-1.044]; T3 = 1.030 [range 1.007-1.055]; P = 0.792). CONCLUSIONS AND RELEVANCE: Our data suggest that factors other than GFR may affect serum SDMA in hyperthyroid cats and that SDMA does not offer an advantage over other biomarkers traditionally used to predict changes in renal function following radioiodine therapy.

Effect of the Probiotic Bacillus subtilis DE-CA9TM on Fecal Scores, Serum Oxidative Stress Markers and Fecal and Serum Metabolome in Healthy Dogs.

BACKGROUND: There is increasing interest in the use of Bacillus species as probiotics since their spore-forming ability favors their survival in the acidic gastric environment over other probiotic species. The subsequent germination of B. subtilis to their vegetative form allows for their growth in the small intestine and may increase their beneficial effect on the host. B. subtilis strains have also previously been shown to have beneficial effects in humans and production animals, however, no reports are available so far on their use in companion animals. STUDY DESIGN: The goal of this study was therefore to investigate the daily administration of 1 × 109 cfu DE-CA9TM orally per day versus placebo on health parameters, fecal scores, fecal microbiome, fecal metabolomics, as well as serum metabolomics and oxidative stress markers in ten healthy Beagle dogs in a parallel, randomized, prospective, placebo-controlled design over a period of 45 days. RESULTS: DE-CA9TM decreased the oxidative status compared to controls for advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS) and reactive oxygen metabolites (d-ROMS), suggesting an antioxidant effect of the treatment. Fecal metabolomics revealed a significant reduction in metabolites associated with tryptophan metabolism in the DE-CA9TM-treated group. DE-CA9TM also significantly decreased phenylalanine and homocysteine and increased homoserine and threonine levels. Amino acid metabolism was also affected in the serum metabolome, with increased levels of urea and cadaverine, and reductions in N-acetylornithine in DE-CA9TM compared to controls. Similarly, changes in essential amino acids were observed, with a significant increase in tryptophan and lysine levels and a decrease in homocysteine. An increase in serum guanine and deoxyuridine was also detected, with a decrease in beta-alanine in the animals that ingested DE-CA9TM. CONCLUSIONS: Data generated throughout this study suggest that the daily administration of 1 × 109 cfu of DE-CA9TM in healthy Beagle dogs is safe and does not affect markers of general health and fecal scores. Furthermore, DE-CA9TM administration had a potential positive effect on some serum markers of oxidative stress, and protein and lipid metabolism in serum and feces.

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.

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.

Differential Transcriptomic Profiles Following Stimulation with Lipopolysaccharide in Intestinal Organoids from Dogs with Inflammatory Bowel Disease and Intestinal Mast Cell Tumor.

Lipopolysaccharide (LPS) is associated with chronic intestinal inflammation and promotes intestinal cancer progression in the gut. While the interplay between LPS and intestinal immune cells has been well-characterized, little is known about LPS and the intestinal epithelium interactions. In this study, we explored the differential effects of LPS on proliferation and the transcriptome in 3D enteroids/colonoids obtained from dogs with naturally occurring gastrointestinal (GI) diseases including inflammatory bowel disease (IBD) and intestinal mast cell tumor. The study objective was to analyze the LPS-induced modulation of signaling pathways involving the intestinal epithelia and contributing to colorectal cancer development in the context of an inflammatory (IBD) or a tumor microenvironment. While LPS incubation resulted in a pro-cancer gene expression pattern and stimulated proliferation of IBD enteroids and colonoids, downregulation of several cancer-associated genes such as Gpatch4, SLC7A1, ATP13A2, and TEX45 was also observed in tumor enteroids. Genes participating in porphyrin metabolism (CP), nucleocytoplasmic transport (EEF1A1), arachidonic acid, and glutathione metabolism (GPX1) exhibited a similar pattern of altered expression between IBD enteroids and IBD colonoids following LPS stimulation. In contrast, genes involved in anion transport, transcription and translation, apoptotic processes, and regulation of adaptive immune responses showed the opposite expression patterns between IBD enteroids and colonoids following LPS treatment. In brief, the crosstalk between LPS/TLR4 signal transduction pathway and several metabolic pathways such as primary bile acid biosynthesis and secretion, peroxisome, renin-angiotensin system, glutathione metabolism, and arachidonic acid pathways may be important in driving chronic intestinal inflammation and intestinal carcinogenesis.

Synbiotic-IgY Therapy Modulates the Mucosal Microbiome and Inflammatory Indices in Dogs with Chronic Inflammatory Enteropathy: A Randomized, Double-Blind, Placebo-Controlled Study.

Chronic inflammatory enteropathy (CE) is a common cause of persistent gastrointestinal signs and intestinal inflammation in dogs. Since evidence links dysbiosis to mucosal inflammation, probiotics, prebiotics, or their combination (synbiotics) may reduce intestinal inflammation and ameliorate dysbiosis in affected dogs. This study's aim was to investigate the effects of the synbiotic-IgY supplement on clinical signs, inflammatory indices, and mucosal microbiota in dogs with CE. Dogs with CE were enrolled in a randomized prospective trial. Twenty-four client-owned dogs were fed a hydrolyzed diet and administered supplement or placebo (diet) for 6 weeks. Dogs were evaluated at diagnosis and 2- and 6-week post-treatment. Outcome measures included clinical activity, endoscopic and histologic scores, inflammatory markers (fecal calprotectin, C-reactive protein), and composition of the mucosal microbiota via FISH. Eleven supplement- and nine placebo-treated dogs completed the trial. After 6 weeks of therapy, clinical activity and endoscopic scores decreased in both groups. Compared to placebo-treated dogs, dogs administered supplement showed decreased calprotectin at 2-week post-treatment, decreased CRP at 2- and 6-week post-treatment increased mucosal Clostridia and Bacteroides and decreased Enterobacteriaceae in colonic biopsies at trial completion. Results suggest a beneficial effect of diet and supplements on host responses and mucosal microbiota in dogs with CE.

Emerging Roles of Urine-Derived Components for the Management of Bladder Cancer: One Man's Trash Is Another Man's Treasure.

Urinary bladder cancer (UBC) is the most common malignancy of the urinary tract in humans, with an estimated global prevalence of 1.1 million cases over 5 years. Because of its high rates of recurrence and resistance to chemotherapy, UBC is one of the most expensive cancers to treat, resulting in significant health care costs. The development of innovative molecular and cellular tools is necessary to refine patient stratification and help predict response to treatment. Urine is an underused resource of biological components shed from bladder tumors, such as exfoliated cells and extracellular vesicles, that could serve as molecular fingerprints and provide valuable biological insights into tumor phenotype and mechanisms of resistance to chemotherapy. Additionally, characterization of urine-derived extracellular vesicles and cells could be used as reliable biomarkers for prediction of response to neoadjuvant therapy.

Harnessing the Biology of Canine Intestinal Organoids to Heighten Understanding of Inflammatory Bowel Disease Pathogenesis and Accelerate Drug Discovery: A One Health Approach.

In a recent issue of the Lancet, the prevalence of Inflammatory Bowel Disease (IBD) was estimated at 7 million worldwide. Overall, the burden of IBD is rising globally, with direct and indirect healthcare costs ranging between $14.6 and $31.6 billion in the U.S. alone in 2014. There is currently no cure for IBD, and up to 40% of patients do not respond to medical therapy. Although the exact determinants of the disease pathophysiology remain unknown, the prevailing hypothesis involves complex interplay among host genetics, the intestinal microenvironment (primarily bacteria and dietary constituents), and the mucosal immune system. Importantly, multiple chronic diseases leading to high morbidity and mortality in modern western societies, including type II diabetes, IBD and colorectal cancer, have epidemiologically been linked to the consumption of high-calorie, low-fiber, high monosaccharide, and high-fat diets (HFD). More specifically, data from our laboratory and others have shown that repeated consumption of HFD triggers dysbiotic changes of the gut microbiome concomitant with a state of chronic intestinal inflammation and increased intestinal permeability. However, progress in our understanding of the effect of dietary interventions on IBD pathogenesis has been hampered by a lack of relevant animal models. Additionally, current in vitro cell culture systems are unable to emulate the in vivo interplay between the gut microbiome and the intestinal epithelium in a realistic and translatable way. There remains, therefore, a critical need to develop translatable in vitro and in vivo models that faithfully recapitulate human gut-specific physiological functions to facilitate detailed mechanistic studies on the impact of dietary interventions on gut homeostasis. While the study of murine models has been pivotal in advancing genetic and cellular discoveries, these animal systems often lack key clinical signs and temporal pathological changes representative of IBD. Specifically, some limitations of the mouse model are associated with the use of genetic knockouts to induce immune deficiency and disease. This is vastly different from the natural course of IBD developing in immunologically competent hosts, as is the case in humans and dogs. Noteworthily, abundant literature suggests that canine and human IBD share common clinical and molecular features, such that preclinical studies in dogs with naturally occurring IBD present an opportunity to further our understanding on disease pathogenesis and streamline the development of new therapeutic strategies. Using a stepwise approach, in vitro mechanistic studies investigating the contribution of dietary interventions to chronic intestinal inflammation and "gut leakiness" could be performed in intestinal organoids and organoid derived monolayers. The biologic potential of organoids stems from the method's ability to harness hard-wired cellular programming such that the complexity of the disease background can be reflected more accurately. Likewise, the effect of therapeutic drug candidates could be evaluated in organoids prior to longitudinal studies in dog and human patients with IBD. In this review, we will discuss the value (and limitations) of intestinal organoids derived from a spontaneous animal disease model of IBD (i.e., the dog), and how it can heighten understanding of the interplay between dietary interventions, the gut microbiota and intestinal inflammation. We will also review how intestinal organoids could be used to streamline the preclinical development of therapeutic drug candidates for IBD patients and their best four-legged friends.

Apparent total tract digestibility, fecal characteristics, and blood parameters of healthy adult dogs fed high-fat diets.

Pet foods may be formulated with decreased starch to meet consumer demands for less processed diets. Fats and oils may be added to low-starch diets to meet energy requirements, but little is known about its effects on canine health. The study objective was to evaluate the effects of feeding healthy adult dogs low carbohydrate, high-fat diets on apparent total tract digestibility, fecal characteristics, and overall health status. Eight adult Beagles were enrolled in a replicated 4 × 4 Latin Square design feeding trial. Dogs were randomly assigned to one of four dietary fat level treatments (T) within each period: 32% (T1), 37% (T2), 42% (T3), and 47% (T4) fat on a dry matter basis. Fat levels were adjusted with the inclusion of canola oil added to a commercial diet. Each dog was fed to exceed its energy requirement based on NRC (2006). Blood samples were analyzed for complete blood counts, chemistry profiles, and canine pancreatic lipase immunoreactivity levels. Apparent total tract digestibility improved (P < 0.05) as the fat level increased for dry matter, organic matter, fat, and gross energy. Fecal output decreased as levels of fat increased in the diet (P = 0.002). There was no effect of fat level on stool quality or short-chain fatty acid and ammonia concentrations in fecal samples (P ≥ 0.20). Blood urea nitrogen levels decreased with increased fat level (P = 0.035). No significant differences were seen in canine pancreatic lipase immunoreactivity (P = 0.110). All blood parameters remained within normal reference intervals. In summary, increased dietary fat improved apparent total tract digestibility, did not alter fecal characteristics, and maintained the health status of all dogs.

CAR T Cell Immunotherapy in Human and Veterinary Oncology: Changing the Odds Against Hematological Malignancies.

The advent of the genome editing era brings forth the promise of adoptive cell transfer using engineered chimeric antigen receptor (CAR) T cells for targeted cancer therapy. CAR T cell immunotherapy is probably one of the most encouraging developments for the treatment of hematological malignancies. In 2017, two CAR T cell therapies were approved by the US Food and Drug Administration: one for the treatment of pediatric acute lymphoblastic leukemia (ALL) and the other for adult patients with advanced lymphomas. However, despite significant progress in the area, CAR T cell therapy is still in its early days and faces significant challenges, including the complexity and costs associated with the technology. B cell lymphoma is the most common hematopoietic cancer in dogs, with an incidence approaching 0.1% and a total of 20-100 cases per 100,000 individuals. It is a widely accepted naturally occurring model for human non-Hodgkin's lymphoma. Current treatment is with combination chemotherapy protocols, which prolong life for less than a year in canines and are associated with severe dose-limiting side effects, such as gastrointestinal and bone marrow toxicity. To date, one canine study generated CAR T cells by transfection of mRNA for CAR domain expression. While this was shown to provide a transient anti-tumor activity, results were modest, indicating that stable, genomic integration of CAR modules is required in order to achieve lasting therapeutic benefit. This commentary summarizes the current state of knowledge on CAR T cell immunotherapy in human medicine and its potential applications in animal health, while discussing the potential of the canine model as a translational system for immuno-oncology research.