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Derivation of adult canine intestinal organoids for translational research in gastroenterology

Large animal models, such as dogs, are increasingly being used to study gastrointestinal (GI) disorders. Dogs share similar environmental, genomic and intestinal physiological features with humans, more so than more commonly used rodent models. The cellular models derived from canine species can offer an excellent alternative to human models in translational research and drug development.

In this study, Chandra et al., have developed a three-dimensional (3D) canine GI organoid model and investigated the characteristics and translational relevance of the model in relation to naturally occurring intestinal diseases such as IBD and cancer in dogs and humans.

Using isolated canine intestine crypts, containing adult intestinal stem cells, the authors developed culture conditions to grow 3D intestinal organoids from various sites of small intestine and colon from 40 (28 healthy and 12 diseased) dogs, including dogs diagnosed with IBD (n=9) and intestinal tumours (n=2).

The 3D cultures could be maintained for up to 20 passages and were successfully cryopreserved and recovered.

Histological assessment of the generated GI organoids showed similarities of epithelial structure between 3D cultures and native gut tissue. Day 3 organoids displayed undifferentiated cyst-like structures, while day 6-9 organoids presented both crypt and villi-like structures characteristic of differentiated tissue.

In depth analysis of organoid cultures by TEM confirmed progressive development (between 3-9 days of differentiation) of inter-epithelial structures, adherens junctions (AJ), thigh junction (TJ) and desmosomes, which are important for intestinal barrier function. The presence of ultrastructural features such as apical microvilli, electron-lucent cytoplasmic vacuoles and electron-dense perinuclear granules were consistent with development of absorptive enterocytes, goblet cells and enteroendocrine cells, respectively, were also detected within the organoid cultures. This analysis confirmed that dog organoid cultures mimic morphologically intact gut tissue.

Similar to the native gut tissue, organoids expressed markers of epithelial cell lineages, including keratin (marker of epithelial cells), Chromogranin A (enteroendocrine cells) and PAS (goblet cells) confirming that those cell types were present within 3D cultures. Paneth cells, which are usually detected by lysosome staining, were absent. In addition, neither immune cells (c-Kit and CD3 positive) nor mesenchymal cells (vimentin and actin positive) were detected in the organoid cultures, while abundant in the native gut tissue.

To characterize intestinal stem cell populations the authors designed a cohort of dog specific RNA in situ hybridization probes and used RNAScope to establish localization and expression levels of stem cell specific RNAs in the 3D cultures. Intestinal stem cells positive for LGR5 and Sox9 were detected mainly at the crypt area, similar to another marker of intestinal stemness EPHB2. In addition, absorptive enterocyte and enteroendocrine cells were detected in canine organoids mostly in villi structures (as marked by intestinal Alkaline Phosphtase (ATP) and Neurogenin-3 (NeuroG3), respectively). The similar distribution of stem and differentiated cells markers were observed within crypt and villi structures of native gut tissue.

In order to investigate whether canine organoids from IBD dogs mimic the molecular signature of the disease of the native IBD gut, the authors examined expression of EP4 prostoglandin receptor (EP4R), which has been previously implicated in pathogenesis of IBD. There was no difference in epithelial expression of EP4R between IBD organoids and IBD biopsies from dogs indicating that canine organoids accurately model whole tissue EPR4 expression and retain their IBD phenotype.

The study also examined functional characteristics of dog organoids including analysis of cellular metabolism between growing and differentiated organoids (by measuring NADH and FAD metabolites and establishing Redox Ratio using OMI technique). They also performed a forskolin-induced swelling of organoids assay as a measure of CFTR function. The results indicated that organoids have active CFTR chloride channels, similar to healthy gut tissue.

Finally, to evaluate the absorptive and barrier functionality of 3D cultures an uptake assay using parasitic EVs produced by intestinal helminths was performed. Exosome-like vesicles from parasites were phagocytized by organoids through the epithelial cells and into the organoid lumen within 24h.

In summary, this study describes the development and long-term maintenance of small intestine and colon in vitro canine organoids using whole tissue and biopsy samples from 40 healthy and diseased dogs. The authors demonstrate that 3D cultures maintain the molecular and functional characteristics of the original gut tissue they were derived from. The organoids complement existing preclinical animal models and provide a robust screening tool for drug permeability, efficacy and drug safety screening to aid the selection of candidate treatments with minimum GI toxicity and maximum efficacy preventing or treating GI diseases.

Reference

Chandra et al., 2019. Derivation of adult canine intestinal organoids for translational research in gastroenterology. BMC Biology, 17:33

https://doi.org/10.1186/s12915-019-0652-6

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Epistem's Gut Organoid Models

Epistem offers validated small intestinal in vitro organoid models derived from variety of species, including human, canine and rodents. Our organoid cultures display cellular architecture that closely resembles that observed in vivo. Cultures mimic the stem cell niche allowing cell proliferation and differentiation to occur. The established organoid protocols allow MOA determination and provide in vitro to in vivo biological and PD linkages through following readouts:

  • Viability assays (visual or with MTS)
  • Histology/IHC or RNAscope to assess the effect on specific protein expression including LGR5
  • Gene expression (NGS, Microarray or qRT-PCR) to evaluate the effect of treatment on expression of signalling pathways genes
  • FACS analysis to identify specific cell populations effected
  • ELISA to identify and quantify cytokines of interest within organoid's spend media.

Epistem's intestinal organoid models allow screening of multiple agents, with over 5000 screened to date including Wnt pathway regulators, cytokines, growth factor, chemotherapy drugs, steroids and lectins.

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