A matter of differentiation: equine enteroids as a model for the in vivo intestinal epithelium.

Epithelial damage due to gastrointestinal disorders frequently causes severe disease in horses. To study the underlying pathophysiological processes, we aimed to establish equine jejunum and colon enteroids (eqJE, eqCE) mimicking the in vivo epithelium. Therefore, enteroids were cultivated in four different media for differentiation and subsequently characterized histomorphologically, on mRNA and on protein level in comparison to the native epithelium of the same donor horses to identify ideal culture conditions for an in vitro model system. With increasing enterocyte differentiation, the enteroids showed a reduced growth rate as well as a predominantly spherical morphology and less budding compared to enteroids in proliferation medium. Combined or individual withdrawal of stem cell niche pathway components resulted in lower mRNA expression levels of stem cell markers and concomitant differentiation of enterocytes, goblet cells and enteroendocrine cells. For eqCE, withdrawal of Wnt alone was sufficient for the generation of differentiated enterocytes with a close resemblance to the in vivo epithelium. Combined removal of Wnt, R-spondin and Noggin and the addition of DAPT stimulated differentiation of eqJE at a similar level as the in vivo epithelium, particularly with regard to enterocytes. In summary, we successfully defined a medium composition that promotes the formation of eqJE and eqCE consisting of multiple cell types and resembling the in vivo epithelium. Our findings emphasize the importance of adapting culture conditions to the respective species and the intestinal segment. This in vitro model will be used to investigate the pathological mechanisms underlying equine gastrointestinal disorders in future studies.

Traces of Canine Inflammatory Bowel Disease Reflected by Intestinal Organoids.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that affects humans and several domestic animal species, including cats and dogs. In this study, we have analyzed duodenal organoids derived from canine IBD patients using quantitative proteomics. Our objective was to investigate whether these organoids show phenotypic traits of the disease compared with control organoids obtained from healthy donors. To this aim, IBD and control organoids were subjected to quantitative proteomics analysis via liquid chromatography-mass spectrometry. The obtained data revealed notable differences between the two groups. The IBD organoids exhibited several alterations at the levels of multiple proteins that are consistent with some known IBD alterations. The observed phenotype in the IBD organoids to some degree mirrors the corresponding intestinal condition, rendering them a compelling approach for investigating the disease and advancing drug exploration. Additionally, our study revealed similarities to some human IBD biomarkers, further emphasizing the translational and comparative value of dogs for future investigations related to the causes and treatment of IBD. Relevant proteins such as CALU, FLNA, MSN and HMGA2, which are related to intestinal diseases, were all upregulated in the IBD duodenal organoids. At the same time, other proteins such as intestinal keratins and the mucosal immunity PIGR were depleted in these IBD organoids. Based on these findings, we propose that these organoids could serve as a valuable tool for evaluating the efficacy of therapeutic interventions against canine IBD.

Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.

Apical-out intestinal organoids are a relatively simple method of gaining access to the apical cell surface and have faced increasing scientific interest over the last few years. Apical-out organoids can thus be used for disease modelling to compare differing effects on the basolateral versus the apical cell surface. However, these 'inside-out' organoids die relatively quickly and cannot be propagated as long as their basal-out counterparts. Here, we show that apical-out organoids have drastically reduced proliferative potential, as evidenced by immunohistochemical staining and the incorporation of the thymidine analogue EdU. At the same time, cell death levels are increased. Nevertheless, these phenomena cannot be explained by an induction of differentiation, as the gene expression of key marker genes for various cell types does not change over time.

Time to eRAASe chronic inflammation: current advances and future perspectives on renin-angiotensin-aldosterone-system and chronic intestinal inflammation in dogs and humans.

Chronic idiopathic intestinal inflammation is an increasing worldwide problem that affects companion animals, especially dogs, and human patients. Although these disease entities have been intensely investigated recently, many questions remain, and alternative therapeutic options are needed. Diarrhea caused by dysregulation of intestinal electrolyte transport and subsequent fluid and electrolyte losses often leads to secondary consequences for the patient. Currently, it is not exactly clear which mechanisms are involved in the dysregulation of intestinal fluid absorption, but differences in intestinal electrolyte shifts between human and canine patients suggest species-specific regulatory or counterregulatory mechanisms. Several intestinal electrolyte transporters are differentially expressed in human patients with inflammatory bowel disease (IBD), whereas there are virtually no studies on electrolyte transporters and their endocrine regulation in canine chronic inflammatory enteropathy. An important mechanism involved in regulating fluid and electrolyte homeostasis is the renin-angiotensin-aldosterone-system (RAAS), which may affect intestinal Na+ transport. While RAAS has previously been considered a systemic regulator of blood pressure, additional complex roles of RAAS in inflammatory processes have been unraveled. These alternative RAAS pathways may pose attractive therapeutic targets to address diarrhea and, thus, electrolyte shifts in human IBD and canine chronic inflammatory enteropathy. This article comparatively summarizes the current knowledge about electrolyte transport in human IBD and canine chronic inflammatory enteropathy and the role of RAAS and offers perspectives for novel therapeutic avenues.

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.

The World of Organoids: Gastrointestinal Disease Modelling in the Age of 3R and One Health with Specific Relevance to Dogs and Cats.

One Health describes the importance of considering humans, animals, and the environment in health research. One Health and the 3R concept, i.e., the replacement, reduction, and refinement of animal experimentation, shape today's research more and more. The development of organoids from many different organs and animals led to the development of highly sophisticated model systems trying to replace animal experiments. Organoids may be used for disease modelling in various ways elucidating the manifold host-pathogen interactions. This review provides an overview of disease modelling approaches using organoids of different kinds with a special focus on animal organoids and gastrointestinal diseases. We also provide an outlook on how the research field of organoids might develop in the coming years and what opportunities organoids hold for in-depth disease modelling and therapeutic interventions.