Organoids as a Model for Intestinal Ion Transport Physiology

The advent of intestinal organoid culture in 2009 was a fortuitous development in the search for a valid marker of intestinal stem cells, and provided proof of murine intestinal stem cell regenerative potential. Intestinal organoid culture was preceded by key discoveries of the Wnt/β-catenin signaling pathway and the development of 3D culture matrices. The latter, involving a laminin-rich gel to provide an artificial basement membrane, was instrumental to primary intestinal epithelial culture by preventing anoikis, an immediate apoptotic event when intestinal epithelial cells detach from the basement membrane. One of the first physiological studies using 3D murine “mini-gut” structures showed cystic fibrosis transmembrane conductance regulator (CFTR) expression and anion channel activity in the crypt-like structures projecting from the epithelial-lined central cavity. Detailed investigations of ion transport physiology using human intestinal organoids, both primary and iPSC-derived, found close similarities to existing knowledge of ion transport physiology and included the development of the forskolin-induced swelling assay (FIS). The FIS assay using organoids cultured from rectal biopsies of cystic fibrosis patients provided an avenue for personalized medicine to test small-molecule modulators on different CFTR mutations. More recent research has led to the development of 2D primary intestinal epithelial monolayers, which provide easy access to the apical, lumen-facing membrane and the opportunity for traditional ion transport studies with Ussing chambers. Human 2D primary intestinal monolayers also demonstrate the dominance of CFTR in anion secretion and provide a quantitative evaluation of its chloride and bicarbonate secretory conductances. These aspects of ion transport physiology using 2D and 3D intestinal cultures are discussed along with the relative advantages and disadvantages of each culture method with respect to technical aspects and recapitulation of native intestinal epithelium

Expression and distribution patterns of Mas-related gene receptor subtypes A-H in the mouse intestine: inflammation-induced changes

Mas-related gene (Mrg) receptors constitute a subfamily of G protein-coupled receptors that are implicated in nociception, and are as such considered potential targets for pain therapies. Furthermore, some Mrgs have been suggested to play roles in the regulation of inflammatory responses to non-immunological activation of mast cells and in mast cell-neuron communication. Except for MrgD, E and F, whose changed expression has been revealed during inflammation in the mouse intestine in our earlier studies, information concerning the remaining cloned mouse Mrg subtypes in the gastrointestinal tract during (patho) physiological conditions is lacking. Therefore, the present study aimed at identifying the presence and putative function of these remaining cloned Mrg subtypes (n = 19) in the (inflamed) mouse intestine. Using reverse transcriptase-PCR, quantitative-PCR and multiple immunofluorescence staining with commercial and newly custom-developed antibodies, we compared the ileum and the related dorsal root ganglia (DRG) of non-inflamed mice with those of two models of intestinal inflammation, i.e., intestinal schistosomiasis and 2,4,6-trinitrobenzene sulfonic acid-induced ileitis. In the non-inflamed ileum and DRG, the majority of the Mrg subtypes examined were sparsely expressed, showing a neuron-specific expression pattern. However, significant changes in the expression patterns of multiple Mrg subtypes were observed in the inflamed ileum; for instance, MrgA4, MrgB2and MrgB8 were expressed in a clearly increased number of enteric sensory neurons and in nerve fibers in the lamina propria, while de novo expression of MrgB10 was observed in enteric sensory neurons and in newly recruited mucosal mast cells (MMCs). The MrgB10 expressing MMCs were found to be in close contact with nerve fibers in the lamina propria. This is the first report on the expression of all cloned Mrg receptor subtypes in the (inflamed) mouse intestine. The observed changes in the expression and cellular localization of the Mrg subtypes suggest that these receptors are involved in the mediation of primary afferent responses, mast cell responses, and in neuroimmune communication during intestinal inflammation