Shiro Yui

Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell

Adult stem-cell therapy holds promise for the treatment of gastrointestinal diseases. Here we describe methods for long-term expansion of colonic stem cells positive for leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5+ cells) in culture. To test the transplantability of these cells, we reintroduced cultured GFP+ colon organoids into superficially damaged mouse colon. The transplanted donor cells readily integrated into the mouse colon, covering the area that lacked epithelium as a result of the introduced damage in recipient mice. At 4 weeks after transplantation, the donor-derived cells constituted a single-layered epithelium, which formed self-renewing crypts that were functionally and histologically normal. Moreover, we observed long-term (>6 months) engraftment with transplantation of organoids derived from a single Lgr5+ colon stem cell after extensive in vitro expansion. These data show the feasibility of colon stem-cell therapy based on the in vitro expansion of a single adult colonic stem cell.

Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury

Summary Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS). A highly similar progenitor population can be established during intestinal differentiation of human induced pluripotent stem cells. Established cultures of mouse fetal intestinal progenitors express lower levels of Lgr5 than mature progenitors and propagate in the presence of the Wnt antagonist Dkk1, and new cultures can be induced to form mature intestinal organoids by exposure to Wnt3a. Following transplantation in a colonic injury model, FEnS contribute to regeneration of colonic epithelium by forming epithelial crypt-like structures expressing region-specific differentiation markers. This work provides insight into mechanisms underlying development of the mammalian intestine and points to future opportunities for patient-specific regeneration of the digestive tract.

YAP/TAZ-Dependent Reprogramming of Colonic Epithelium Links ECM Remodeling to Tissue Regeneration

Summary Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.

Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells.

Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

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.

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells.

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.

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.

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3- or Cl-. Bestrophin genes represent a newly identified group of calcium-activated Cl- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

Single cell analysis of Crohn’s disease patient-derived small intestinal organoids reveals disease activity-dependent modification of stem cell properties

BackgroundIntestinal stem cells (ISCs) play indispensable roles in the maintenance of homeostasis, and also in the regeneration of the damaged intestinal epithelia. However, whether the inflammatory environment of Crohn’s disease (CD) affects properties of resident small intestinal stem cells remain uncertain.MethodsCD patient-derived small intestinal organoids were established from enteroscopic biopsy specimens taken from active lesions (aCD-SIO), or from mucosa under remission (rCD-SIO). Expression of ISC-marker genes in those organoids was examined by immunohistochemistry, and also by microfluid-based single-cell multiplex gene expression analysis. The ISC-specific function of organoid cells was evaluated using a single-cell organoid reformation assay.ResultsISC-marker genes, OLFM4 and SLC12A2, were expressed by an increased number of small intestinal epithelial cells in the active lesion of CD. aCD-SIOs, rCD-SIOs or those of non-IBD controls (NI-SIOs) were successfully established from 9 patients. Immunohistochemistry showed a comparable level of OLFM4 and SLC12A2 expression in all organoids. Single-cell gene expression data of 12 ISC-markers were acquired from a total of 1215 cells. t-distributed stochastic neighbor embedding analysis identified clusters of candidate ISCs, and also revealed a distinct expression pattern of SMOC2 and LGR5 in ISC-cluster classified cells derived from aCD-SIOs. Single-cell organoid reformation assays showed significantly higher reformation efficiency by the cells of the aCD-SIOs compared with that of cells from NI-SIOs.ConclusionsaCD-SIOs harbor ISCs with modified marker expression profiles, and also with high organoid reformation ability. Results suggest modification of small intestinal stem cell properties by unidentified factors in the inflammatory environment of CD.

26 The Cellular Plasticity of Fetal and Adult Intestinal Epithelium and Its Functional Evaluation by Grafting Assay

histology), circularity values increased up to 0.9. C57BL/6 enteroids showed a dose dependent increase in circularity in response to TNF (10-1000ng/ml) or IFN γ (1-100ng/ml) after 24 and 48 hours. Circularity did not however increase in Nfkb2-/enteroids to the same extent following these stimuli. C57BL/6 BMDCs or their CM or LPS alone did not induce an increase in the circularity of C57BL/6, Nfkb2 or Nfkb1-/enteroids. However, when stimulated with 1μg/ml LPS, C57BL/6 BMDCs and their CM induced a significant >2-fold increase in the circularity of C57BL/6 and Nfkb1-/enteroids. Nfkb2 derived enteroids showed no such increase. LPS stimulated BMDCs from Nfkb2-/mice were able to induce a significant increase in C57BL/6 enteroid circularity of a similar magnitude to C57BL/6 BMDCs. Conclusions:Circularity correlates well with cell death and shedding in murine enteroid culture. Deletion of Nfkb2 from intestinal epithelial cells prevents epithelial cell destruction in response to secreted immune cell factors. Therapeutics that specifically target NFKB2 signalling in intestinal epithelial cells may therefore prevent loss of intestinal barrier function and thus ameliorate intestinal inflammation.