Organoids as an ex vivo model for studying the serotonin system in the murine small intestine and colon epithelium
Introduction
Although the intestinal epithelium has been difficult to model in culture, the establishment of a system for culturing primary stem cell–derived intestinal organoids has overcome this difficulty [1], [2], [3]. Intestinal organoids can be produced from isolated intestinal crypts that include stem cells by three-dimensional Matrigel culturing in medium containing a combination of Wnt, R-spondin, and Noggin [1], [2], [3]. Small intestinal organoids consist of a polarized epithelium that is patterned into villus-like regions containing differentiated enterocytes, goblet cells, and enteroendocrine (EE) cells and crypt-like proliferative zones containing stem cells, transit-amplifying cells, and Paneth cells [1]. Thus, small intestinal organoids recapitulate critical in vivo characteristics, such as the cellular composition and self-renewal kinetics of the small intestine epithelium [1].
Enterochromaffin (EC) cells are a subset of EE cells and are responsible for the synthesis, storage, and release of serotonin (5-hydroxytryptamine, 5-HT), a sensory mediator in the intestine (for reviews, see Refs. [4] and [5]). 5-HT is released from EC cells in response to chemical and mechanical stimuli in the intestinal lumen and mediates many intestinal functions, including peristalsis, secretion, and vasodilation, through activation of a diverse family of 5-HT receptors (HTRs) on intrinsic and extrinsic sensory neurons located in the lamina propria of the intestinal mucosa. The actions of 5-HT are terminated by uptake via the serotonin reuptake transporter (SERT) into intestinal epithelial cells. 5-HT can also act directly on epithelial cells via HTR2 [6] and HTR4 [7], [8]. Thus, intestinal epithelial cells play a central role in the 5-HT system (i.e., in the synthesis, release, reuptake, and recognition of 5-HT). The present study aimed to test whether intestinal organoids are useful for studying the 5-HT system in the intestinal epithelium.
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Animal care
Female C57BL/6N mice (age 5 weeks) were purchased from Japan SLC and housed in standard plastic cages in a temperature-controlled (23 ± 2 °C) room under a 12-h light/12-h dark cycle and were allowed free access to tap water and standard laboratory rodent feed (Oriental Yeast). All study protocols were approved by the Animal Use Committee of Hokkaido University (approval no. 08–0139). Animals were maintained in accordance with the Hokkaido University guidelines for the care and use of laboratory
Small intestinal and colonic organoids express 5-HT–related genes
We successfully cultured murine small intestinal and colonic organoids (Fig. 1A). On day 5 of culture, we observed the typical structure of mature intestinal organoids, consisting of a central cyst structure and surrounding crypt-like budding structures in both the small intestine and colon. Organoids were used for the experiments on day 6 of culture. Ethidium bromide staining of PCR products separated by electrophoresis on a 2% agarose gel showed that both the small intestinal and colonic
Discussion
The present study examined the expression of mRNAs encoding 5-HT–related proteins in intestinal mucosal tissues and organoids. We found that TPH1 mRNA is expressed in both the small intestinal and colonic organoids as well as the mucosal tissues, suggesting that epithelial cells express TPH1 mRNA. This result is reasonable, because EC cells that express the TPH1 mRNA are a subset of EE cells located in the intestinal epithelium. In the same manner, our data suggest that SERT mRNA is expressed
Conflict of interest
The authors have no conflict of interest to disclose.
Acknowledgments
This study was supported in part by the Regional Innovation Strategy Support Program of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), by the Center of Innovation Program from the MEXT and Japan Science and Technology Agency, and by the Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP) “Technologies for creating next-generation agriculture, forestry and fisheries”. The authors have no conflicts of
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These authors contributed equally to this work.