Organoids: Key to understanding gut health?

B. van der Hee*, M.S. Gilbert, W.J.J. Gerrits, H. Smidt, J.M. Wells

*Corresponding author for this work

Research output: Contribution to conferencePosterProfessional

Abstract

In Dutch farming, piglets are commonly weaned at around 28 days of age, where this process is usually abrupt. This abrupt separation from the sow is often associated with stress-induced pathology, including malnutrition, intestinal morphological changes, and/or aberrations in microbiota composition and activity. Furthermore, the transition from sow’s milk to solid feed leads to worse digestion of dietary components, such as protein, in the small intestine, leading to a higher flow of undigested nutrients into the colon. Subsequently, these could be fermented by proteolytic bacterial species. The metabolites of this protein fermentation have been associated with toxic or pro-inflammatory impact on the host epithelium. Corroborating this hypothesis, studies have shown that feeding piglets with a diet high in protein content is associated with higher incidence of diarrhea, and that decreasing protein content often leads to alleviation of the problems. However, not all studies have shown the same results regarding the relation between diarrhea and protein content when factoring in protein contents normally used in animal production, nor the presence or absence of protein degrading bacteria.
Initial results of our pilot and on-farm studies have shown a strong negative correlation between ammonia concentration and dry matter content of the feces. This indicates that there is an association between ammonia, a metabolite produced during protein fermentation, and diarrhea. As we are interested in the underlying mechanisms including interactions between luminal content and the host epithelium, we set out to study intestinal responses to protein fermentation metabolites in the lab using in vitro models. Previously, such models used transformed or cancerous tissue derived cells. Despite being a powerful tool for many decades, these models often undergo chromosomal aberrations or mutations and are often difficult to translate to in vivo host responses. In 2009, researchers from Utrecht University, The Netherlands, and simultaneously at Stanford University, U.S.A., a model was developed by growing intestinal stem cells in a 3-dimensional matrix to form organ-resembling structures called organoids [1, 2]. In our lab, we have employed these techniques to form intestinal organoids from pigs of various intestinal locations, that represent specific host genetics and cell types.
These 3-dimensional porcine intestinal structures could be dissociated and plated to form 2-dimensional monolayers, still retaining a basic host and epithelial phenotype [3]. This enables us to grow single-layer epithelia in high-throughput format to study fundamental aspects of interactions between host tissues and luminal compounds. One of our interests is defining the effects different metabolites have on the host epithelial barrier. One way of measuring this is by mimicking wound-healing abilities of the cell monolayer. Intestinal health can be defined by the ability of the barrier to regain or retain homeostasis when environmental conditions change [4]. Barrier integrity is essential for separating luminal content from the host, where dysbiosis often leads to a cascade of multiple associated illnesses, like diarrhea. When the barrier is compromised, this needs to be resolved acutely through a process called re-epithelialization to further prevent infections.
Our intestinal monolayer model can be used to mimic epithelial damage and follow the kinetics of wound healing over time [5]. Following protocols developed in our lab, we can induce a reproducible scratch along the epithelium, and follow cellular infiltration over time (Figure 1a). By staining the nuclei of these cells (Figure 1b), we can use image analysis to identify cells entering the lesion by computational methods (Figure 1c) and calculate re-epithelialization kinetics over time (Figure 1d). This model can be used to identify the rate of wound repair, and the effects various compounds can have on this process [6]. Since kinetics of wound healing by inducing cellular stress can often be more informative than stable cell layers, this might provide novel insight into cell migration and proliferation upon treatment with protein fermentation metabolites, diarrheal extracts, or (un)digested feed components.

References
1. Ootani, A., et al., Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nature Medicine, 2009. 15(6): p. 1-U140.
2. Sato, T., et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 2009. 459(7244): p. 262-U147.
3. van der Hee, B., et al., Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids. Stem Cell Research, 2018. 28: p. 165-171.
4. Bischoff, S.C., et al., Intestinal permeability--a new target for disease prevention and therapy. BMC Gastroenterol, 2014. 14: p. 189.
5. Fernandez-Gutierrez, M.M., et al., Streptococcus salivarius MS-oral-D6 promotes gingival reepithelialization in vitro through a secreted serine protease. Scientific Reports, 2017. 7.
6. Fernandez-Gutierrez, M.M., et al., KREAP: an automated Galaxy platform to quantify in vitro re-epithelialization kinetics. Gigascience, 2018. 7(7).

Original languageEnglish
Pages1-2
Number of pages2
Publication statusPublished - 5 Nov 2018
Event2nd annual meeting of STW-DSM program: DSM HANS - Hannover, Hannover, Germany
Duration: 5 Nov 20187 Nov 2018

Conference

Conference2nd annual meeting of STW-DSM program
CountryGermany
CityHannover
Period5/11/187/11/18

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