Xiu Zheng

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.

Suppression of hath1 gene expression directly regulated by hes1 via notch signaling is associated with goblet cell depletion in ulcerative colitis

Background: The transcription factor Atoh1/Hath1 plays crucial roles in the differentiation program of human intestinal epithelium cells (IECs). Although previous studies have indicated that the Notch signal suppresses the differentiation program of IEC, the mechanism by which it does so remains unknown. This study shows that the undifferentiated state is maintained by the suppression of the Hath1 gene in human intestine. Methods: To assess the effect of Notch signaling, doxycycline‐induced expression of Notch intracellular domain (NICD) and Hes1 cells were generated in LS174T. Hath1 gene expression was analyzed by quantitative reverse‐transcription polymerase chain reaction (RT‐PCR). Hath1 promoter region targeted by HES1 was determined by both reporter analysis and ChIP assay. Expression of Hath1 protein in ulcerative colitis (UC) was examined by immunohistochemistry. Results: Hath1 mRNA expression was increased by Notch signal inhibition. However, Hath1 expression was suppressed by ectopic HES1 expression alone even under Notch signal inhibition. Suppression of the Hath1 gene by Hes1, which binds to the 5′ promoter region of Hath1, resulted in suppression of the phenotypic gene expression for goblet cells. In UC, the cooperation of aberrant expression of HES1 and the disappearance of caudal type homeobox 2 (CDX2) caused Hath1 suppression, resulting in goblet cell depletion. Conclusions: The present study suggests that Hes1 is essential for Hath1 gene suppression via Notch signaling. Moreover, the suppression of Hath1 is associated with goblet cell depletion in UC. Understanding the regulation of goblet cell depletion may lead to the development of new therapy for UC. (Inflamm Bowel Dis 2011;)

The acquisition of malignant potential in colon cancer is regulated by the stabilization of Atonal homolog 1 protein

The transcription factor Atonal homolog 1 (Atoh1) plays crucial roles in the differentiation of intestinal epithelium cells. Although we have reported that the Atoh1 protein was degraded in colon cancer by aberrant Wnt signaling, a recent study has indicated that the Atoh1 protein is expressed in mucinous colon cancer (MC) and signet ring cell carcinoma (SRCC). However, the roles of the Atoh1 protein in MC are unknown. To mimic MC, a mutated Atoh1 protein was stably expressed in undifferentiated colon cancer cells. Microarray analysis revealed the acquisition of not only the differentiated cell form, but also malignant potential by Atoh1 protein stabilization. In particular, Atoh1 enhanced Wnt signaling, resulting in the induction of Lgr5 as a representative stem cell marker with the enrichment of cancer stem cells. Moreover, the fluorescent ubiquitination-based cell cycle indicator system with time-lapse live imaging demonstrated cell cycle arrest in the G0/G1 phase by Atoh1 protein stabilization. In conclusion, the Atoh1 protein regulates malignant potential rather than the differentiation phenotype of MC, suggesting the mechanism by which MC and SRCC are more malignant than non-mucinous adenocarcinoma.

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.

Su1731 Stabilization of ATOH1 Protein by TNF-α in Colitic Cancer Might Acquire the Cancer Stemness

ine treated BxPc3 and Hpac cell lines compared to pre-treatment cell lines. CD24+CD44+ cells sorted from the gemcitabine treated cell lines showed higher migration and invasion ability compared to CD24-CD44cells from the same cell lines. Western blot showed the expression of Hes1, pAKT, and beta-catenin was increased in gemcitabine treated cell lines. The overall survival of pancreatic cancer patients with strong expression of Hes1 was shorter than patients with none or weak expression (21.6 vs 11.1 months p=0.036). Treatment with DAPT reversed the increase of Hes1, beta-catenin expression and CD24+CD44+ cells, and also decreased the migration and invasion ability of gemcitabine treated cell lines. CONCLUSION: Notch pathway was involved in the increase and activation of CSC, which might lead to the treatment failure of pancreatic cancer. Notch pathway is a potential treatment target to improve the survival of pancreatic cancer patients.

Su1736 Live Imaging of Single Cell Reveals Single Stem Cell Dynamics in an Organoid Derived From Murine Small Intestine

G A A b st ra ct s differentiation, thus showing that in analogy to the intestine, esophageal stemness is lost. Using RNA micro-arrays, we identified a gene signature of 47 genes that were lost in both individual cell lines upon induction of ER stress. Of these genes, we found 29 genes to be restricted to the basal layer of the mouse esophagus. Out of these 29, nine genes show expression in only a small proportion of the basal cells, potentially marking stem cells. Conclusion: ER stress depletes esophageal precursor cells. Our in vitro screen combined with in situ hybridization identified nine genes that are specifically expressed in a subset of proliferating genes, thereby potentially marking esophageal stem cells.

T1733 The Contribution of Atoh1/Hath1 to Phenotypic Gene Expression for Paneth Cells

with this activity. A combination of four subunits reached highest glucogenesis indicating a broader spectrum of glucosidase activities than any individual subunit alone. Some alphaLDx was not completely available for enzyme hydrolysis, and residues are considered either slowly digestible or resistant to human enzyme digestion. This study for the first time shows the direct role of individual mucosal glucosidase subunits in starch digestion and reveals the potential of producing slow glucose release dextrins.