Masayoshi Fukuda

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.

Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt

BACKGROUND AND AIMS The dynamics of intestinal stem cells are crucial for regulation of intestinal function and maintenance. Although crypt stem cells have been identified in the intestine by genetic marking methods, identification of plural crypt stem cells has not yet been achieved as they are visualised in the same colour. METHODS Intestinal organoids were transferred into Matrigel® mixed with lentivirus encoding mCherry. The dynamics of mCherry-positive cells was analysed using time-lapse imaging, and the localisation of mCherry-positive cells was analysed using 3D immunofluorescence. RESULTS We established an original method for the introduction of a transgene into an organoid generated from mouse small intestine that resulted in continuous fluorescence of the mCherry protein in a portion of organoid cells. Three-dimensional analysis using confocal microscopy showed a single mCherry-positive cell in an organoid crypt that had been cultured for >1year, which suggested the presence of long-lived mCherry-positive and -negative stem cells in the same crypt. Moreover, a single mCherry-positive stem cell in a crypt gave rise to both crypt base columnar cells and transit amplifying cells. Each mCherry-positive and -negative cell contributed to the generation of organoids. CONCLUSIONS The use of our original lentiviral transgene system to mark individual organoid crypt stem cells showed that long-lived plural crypt stem cells might independently serve as intestinal epithelial cells, resulting in the formation of a completely functional villus.

Efficacy of Endocuff-assisted colonoscopy in the detection of colorectal polyps

Background and study aims  Colonoscopy is the gold standard for detecting colorectal adenomas and cancers. Endoscopic surveillance has been shown to be effective for preventing colorectal cancer. Although detection of colorectal polyps at an early stage is important, endoscopic visualization of early neoplasia can be difficult. The Endocuff is a new device that can be attached to the tip of the colonoscope to hold the colonic folds away from the field of view during withdrawal. The aim of this study was to compare the adenoma detection rate (ADR) and the mean number of adenomas detected per patient (MAP) achieved using Endocuff-assisted colonoscopy (EAC) and standard colonoscopy (SC). Patients and methods  This randomized prospective study was conducted at two academic endoscopy departments in Japan. A total of 447 patients underwent a complete colonoscopic examination between April 2015 and September 2015. The EAC group included 239 patients. The cecal intubation rate, insertion time, withdrawal time, pain score, complications, polyp detection rate (PDR), ADR, the mean number of polyps detected per patient (MPP), and the MAP were assessed. Results  There were no differences between the EAC and SC groups in terms of cecal intubation rate, insertion time, withdrawal time, or pain scores. The PDR in patients increased by about 12 % (61.9 % vs. 49.2 %, P  = 0.013) and ADR increased by 15 % (52.5 % vs. 39.2 %, P  = 0.001) with the use of the Endocuff. The advanced ADR was higher in the EAC group but no statistically significant difference was found (7.7 % vs. 4.6 %, P  = 0.17). Both MPP and MAP were also higher in the EAC group (mean ± SD: 1.33 ± 1.43 vs. 0.83 ± 0.99 per patient; P  < 0.01, 1.11 ± 1.41 vs. 0.66 ± 0.99 per patient; P  < 0.01, respectively). No major complications occurred. Conclusions  EAC not only enabled a higher ADR but also significantly increased the mean number of adenomas identified per patient, as compared with SC.

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.

203 Successful Engraftment of Cultured Small Intestinal Epithelial Stem Cells Onto Damaged Colonic Mucosa by Heterotopic Transplantation

were established and demonstrate that rID existed in proximal and distal human tissues and maintained in the enteroids. These data support the observation that rID is maintained cell autonomously by ISCs, independent of input from the surrounding niche. Therefore, the rID is enforced by differential chromatin marks suggesting that an epigenomic state maintains the rID within ISCs. This study provides novel insight towards understanding regional differences within ISCs that will be informative to both basic science and clinical intestinal research.