The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Abstract

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

Defining the early stages of intestinal colonisation by whipworms

“…To our knowledge, our caecaloid-whipworm larvae model is the first example of a live metazoan parasite infecting organoids to establish complex interactions with host cells. While this manuscript was in revision, Smith et al showed that L3 larvae of Teladorsagia circumcinta can invade 3D ovine abomasum and ileum organoids, but by burrowing through Matrigel domes, and passing through the basal side of the epithelium to reach the organoid lumen 36 . In our system, key aspects of the relationship between pathogen and host were maintained.…”

Advancing animal health and disease research in the lab with three‐dimensional cell culture systems

“…The types of 3D cell culture systems that have been developed for livestock and companion animals to date reflects tissues that are commonly infected in these animals, and where there is a pressing need to better understand certain diseases processes. For example, organoids representing GI tissues have been most readily applied in new models of host–pathogen interactions given that many pathogens colonise or gain entry via the GI tract, and there are now published examples of GI organoids derived from each of the major companion and livestock animal species 12,13,17,27,30–32 …”

“…The resulting organoid will consist of various epithelial cell types associated with this particular tissue, including enterocytes, mucous-producing cells, paneth cells, enteroendocrine cells, tuft cells and transit amplifying cells. 12,13 Provided the organoids retain a stem cell subpopulation among their multicellular cell types, they will continue to propagate, allowing continuous cultivation across multiple passages in vitro.…”

“…We have shown that GI stem cells collected from the abomasum (gastric system) and ileum (small intestinal system) of sheep form distinctly different, tissue-specific organoids even when grown under identical conditions. 13 Similarly, organoids derived from different sections of the GI system of humans and mice have been shown to respond differently to microbial stimuli, with innate responses in small intestine organoids differing from those of stomach or colon organoids. 20 Furthermore, even closely related tissue sites can produce distinctly different organoid populations, as shown in a recent study of human biliary tissues.…”

“…When generating organoids from primary stem cells derived from animal tissue, it is important to consider certain factors, including animal age and the specific site of tissue collection. We have shown that GI stem cells collected from the abomasum (gastric system) and ileum (small intestinal system) of sheep form distinctly different, tissue‐specific organoids even when grown under identical conditions 13 . Similarly, organoids derived from different sections of the GI system of humans and mice have been shown to respond differently to microbial stimuli, with innate responses in small intestine organoids differing from those of stomach or colon organoids 20 .…”