Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon.

To develop stem cell therapy for small intestinal (SI) diseases, it is essential to determine whether SI stem cells in culture retain their tissue regeneration capabilities. By using a heterotopic transplantation approach, we show that cultured murine SI epithelial organoids are able to reconstitute self-renewing epithelia in the colon. When stably integrated, the SI-derived grafts show many features unique only to the SI but distinct from the colonic epithelium. Our study provides evidence that cultured adult SI stem cells could be a source for cell therapy of intestinal diseases, maintaining their identity along the gastrointestinal tract through an epithelium-intrinsic mechanism.

Real-time analysis of P-glycoprotein-mediated drug transport across primary intestinal epithelium three-dimensionally cultured in vitro.

P-glycoprotein (P-gp) is an efflux transporter that regulates bioavailability of orally administered drugs at the intestinal epithelium. To develop an in vitro experimental model that mimics P-gp-mediated intestinal drug transport in vivo, we employed normal intestinal epithelium three-dimensionally cultured. Physiological expression of P-gp mRNA and the expression of its protein at the apical membrane were observed in the small intestinal epithelium grown as cystic organoids. Rhodamine123 (Rh123), a substrate for P-gp, was actively transported in the basoapical direction and accumulated in the luminal space, while the epithelial integrity was kept intact. Furthermore, we were able to monitor the whole process of Rh123 transport and its inhibition by verapamil in real-time, from which kinetic parameters for Rh123 transport could be estimated by a mathematical modeling. The method here described to evaluate the dynamics of P-gp-mediated transport in primary intestinal epithelial cells would be instrumental in investigating the physiological function of P-gp and its inhibitors/inducers in vitro.

Distinct intestinal adaptation for vitamin B12 and bile acid absorption revealed in a new mouse model of massive ileocecal resection.

Ileocecal resection (ICR), one of several types of intestinal resection that results in short bowel syndrome (SBS), causes severe clinical disease in humans. We here describe a mouse model of massive ICR in which 75% of the distal small intestine is removed. We demonstrate that mice underwent 75% ICR show severe clinical signs and high mortality, which may recapitulate severe forms of human SBS, despite an adaptive response throughout the remnant intestine. By using this model, we also investigated whether the epithelium of the remnant intestine shows enhanced expression of factors involved in region-specific functions of the ileum. Cubn mRNA and its protein product, which play an essential role in vitamin B12 absorption in the ileum, are not compensatory up-regulated in any part of the remnant intestine, demonstrating a clear contrast with post-operative up-regulation of genes involved in bile acid absorption. Our study suggests that functional adaptation by phenotypical changes in the intestinal epithelium is not a general feature for nutrient absorption systems that are confined to the ileum. We also propose that the mouse model developed in this study will become a unique system to facilitate studies on SBS with ICR in humans.

CCN3 Expression Marks a Sulfomucin-nonproducing Unique Subset of Colonic Goblet Cells in Mice.

Intestinal goblet cells are characterized by their unique morphology and specialized function to secrete mucins. Although it is known that they are a heterogeneous population of cells, there have been few studies that relate the expression of a particular gene with functionally distinct subpopulations of intestinal goblet cells. Here we show that CCN3, a gene encoding a member of the CCN family proteins, is induced by inhibition of Notch signaling in colonic epithelial cells and expressed in goblet cells in mice. We demonstrate that CCN3 expression is confined to a subpopulation of goblet cells in the lower crypt of the proximal and middle colon. In addition, CCN3+ cells in the colon correlate well with the cells that are positive for alcian blue (AB) staining but negative for high-iron diamine (HID) staining in histology. We also show that CCN3+ cells, which are absent in the normal distal colon, transiently and ectopically emerge in regenerating crypts during the repair phase of DSS-induced colitis model. Our study thus suggests that CCN3 labels a unique subpopulation of sulfomucin-nonproducing colonic goblet cells that function in both normal and diseased colonic epithelia.

Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes.

BACKGROUND: Intraepithelial lymphocytes (IELs) in the intestine play important roles in the regulation of local immune responses. Although their functions have been studied in a variety of animal experiments, in vitro studies on spatiotemporal behaviors of IELs and their interaction with intestinal epithelial cells (IECs) have been hampered due to the lack of a suitable culture system. In this study, we aimed at developing a novel co-culture system of IELs with IECs to investigate dynamic interaction between these two populations of cells in vitro. METHODS: We optimized experimental conditions under which murine IELs can be efficiently maintained with IECs cultured as three-dimensional organoids. We then tested the effect of IL-2, IL-7, and IL-15 on the maintenance of IELs in this co-culture system. By time-lapse imaging, we also examined the dynamic behaviors of IELs. RESULTS: IELs can be expanded with epithelial organoids in the presence of IL-2, IL-7, and IL-15. IELs were efficiently maintained within and outside of organoids showing a ~four-fold increase in both αßT and γδT IELs for a period of 2 weeks. Four-dimensional fluorescent imaging revealed an active, multi-directional movement of IELs along the basolateral surface of IECs, and also their inward or outward migration relative to organoid structures. Cell tracking analysis showed that αßT and γδT IELs shared indistinguishable features with regard to their dynamics. CONCLUSIONS: This novel co-culture method could serve as a unique tool to investigate the motility dynamics of IELs and their temporal and spatial interaction with IECs in vitro.

Retinol Promotes In Vitro Growth of Proximal Colon Organoids through a Retinoic Acid-Independent Mechanism.

Retinol (ROL), the alcohol form of vitamin A, is known to control cell fate decision of various types of stem cells in the form of its active metabolite, retinoic acid (RA). However, little is known about whether ROL has regulatory effects on colonic stem cells. We examined in this study the effect of ROL on the growth of murine normal colonic cells cultured as organoids. As genes involved in RA synthesis from ROL were differentially expressed along the length of the colon, we tested the effect of ROL on proximal and distal colon organoids separately. We found that organoid forming efficiency and the expression level of Lgr5, a marker gene for colonic stem cells were significantly enhanced by ROL in the proximal colon organoids, but not in the distal ones. Interestingly, neither retinaldehyde (RAL), an intermediate product of the ROL-RA pathway, nor RA exhibited growth promoting effects on the proximal colon organoids, suggesting that ROL-dependent growth enhancement in organoids involves an RA-independent mechanism. This was confirmed by the observation that an inhibitor for RA-mediated gene transcription did not abrogate the effect of ROL on organoids. This novel role of ROL in stem cell maintenance in the proximal colon provides insights into the mechanism of region-specific regulation for colonic stem cell maintenance.