Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium

Snail family members regulate epithelial‐to‐mesenchymal transition (EMT) during invasion of intestinal tumours, but their role in normal intestinal homeostasis is unknown. Studies in breast and skin epithelia indicate that Snail proteins promote an undifferentiated state. Here, we demonstrate that conditional knockout of Snai1 in the intestinal epithelium results in apoptotic loss of crypt base columnar stem cells and bias towards differentiation of secretory lineages. In vitro organoid cultures derived from Snai1 conditional knockout mice also undergo apoptosis when Snai1 is deleted. Conversely, ectopic expression of Snai1 in the intestinal epithelium in vivo results in the expansion of the crypt base columnar cell pool and a decrease in secretory enteroendocrine and Paneth cells. Following conditional deletion of Snai1, the intestinal epithelium fails to produce a proliferative response following radiation‐induced damage indicating a fundamental requirement for Snai1 in epithelial regeneration. These results demonstrate that Snai1 is required for regulation of lineage choice, maintenance of CBC stem cells and regeneration of the intestinal epithelium following damage.     

哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

哺乳动物肠上皮屏障中非编码RNAs的调控作用

哺乳动物肠上皮是一种拥有快速自我更新能力的组织,在维持机体免疫稳态与肠道应激后的损伤修复中发挥重要作用。源于隐窝底部的多能肠干细胞不断进行增殖、迁移与分化,并沿隐窝 绒毛轴向上移动,从而维持肠上皮完整性。该过程受严格而复杂的基因调控网络参与。越来越多的数据表明,肠上皮完整性受到广泛的非编码RNA的调控,主要包括肠黏膜再生、保护与上皮屏障功能等方面。本文重点讨论了两类非编码RNA(包括microRNAs和lncRNAs)转录后调控肠上皮屏障功能的研究进展。其中,miR-503、miR-146和lnc-uc.173、lnc-SPRY4-IT1、lnc-plncRNA1、lnc-Gata6等,能够促进肠黏膜的更新,增强上皮屏障功能;相反,miR-222、miR-29b、miR-195和lnc-H19与lnc-BC012900等,抑制肠上皮再生并破坏肠上皮屏障功能。miRNAs、mRNAs与lncRNAs间构成复杂的分子网络,共同调控肠上皮稳态。深入研究与肠上皮相关的miRNAs和IncRNAs分子及其作用机制,探寻引起肠黏膜炎症的关键分子靶标,为肠道炎症临床诊治提供新方向与新方法。     

Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

Intestinal organoids were established as an ex vivo model of the intestinal epithelium. We investigated whether organoids resemble the intestinal epithelium in their microRNA (miRNA) profiles. Total RNA samples were obtained from crypt and villus fractions in murine intestine and from cultured organoids. Microarray analysis showed that organoids largely resembled intestinal epithelial cells in their miRNA profiles. In silico prediction followed by qRT-PCR suggested that six genes are regulated by corresponding miRNAs along the crypt-villus axis, suggesting miRNA regulation of epithelial cell renewal in the intestine. However, such expression patterns of miRNAs and their target mRNAs were not reproduced during organoids maturation. This might be due to lack of luminal factors and endocrine, nervous, and immune systems in organoids and different cell populations between in vivo epithelium and organoids. Nevertheless, we propose that intestinal organoids provide a useful in vitro model to investigate miRNA expression in intestinal epithelial cells.     

Toll‐like receptor 2‐mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome‐associated transcellular transcytosis

Peptidoglycan (PGN) is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb PGN from the lumen. Nonetheless, how PGN is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized PGN transport in vitro using IEC‐18 and HT29‐CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual‐labeled PGN by enterocytes. Engulfed PGN was found to form a complex with PGN recognition protein‐3, which may facilitate delivering PGN in vivo. Utilizing electronic microscopy, we revealed that uptake of apical PGN across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of PGN using the transwell system. Our data indicated that apically loaded PGN was exocytosed to the basolateral compartment with exosomes by HT29‐CL19A cells. The PGN‐contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled PGN, we found that luminal PGN was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre‐confluent immature but not post‐confluent mature C2BBe1 cells engulfed PGN via a toll‐like receptor 2‐dependent manner. Together, our findings suggest that (1) crypt‐based immature intestinal epithelial cells play an important role in transport of luminal PGN over the intestinal epithelium; and (2) luminal PGN is transcytosed across intestinal epithelia via a toll‐like receptor 2‐mediated phagocytosis‐multivesicular body‐exosome pathway. The absorbed PGN and its derivatives may facilitate maintenance of intestinal immune homeostasis. J. Cell. Physiol. 222: 658–668, 2010. © 2009 Wiley‐Liss, Inc.     

Integrin α8β1 regulates adhesion,migration and proliferation of human intestinal crypt cells via a predominant RhoA/ROCK‐dependent mechanism

Background. Integrins are transmembrane αβ heterodimer receptors that function as structural and functional bridges between the cytoskeleton and ECM (extracellular matrix) molecules. The RGD (arginine‐glycine‐aspartate tripeptide motif)‐dependent integrin α8β1 has been shown to be involved in various cell functions in neuronal and mesenchymal‐derived cell types. Its role in epithelial cells remains unknown. Results. Integrin α8β1 was found to be expressed in the crypt cell population of the human intestine but was absent from differentiating and mature epithelial cells of the villus. The function of α8β1 in epithelial crypt cells was investigated at the cellular level using normal HIECs (human intestinal epithelial cells). Specific knockdown of α8 subunit expression using an shRNA (small‐hairpin RNA) approach showed that α8β1 plays important roles in RGD‐dependent cell adhesion, migration and proliferation via a RhoA/ROCK (Rho‐associated kinase)‐dependent mechanism as demonstrated by active RhoA quantification and pharmacological inhibition of ROCK. Moreover, loss of α8β1, through RhoA/ROCK, impairs FA (focal adhesion) complex integrity as demonstrated by faulty vinculin recruitment. Conclusions. Integrin α8β1 is expressed in epithelial cells. In intestinal crypt cells, α8β1 is closely involved in the regulation of adhesion, migration and cell proliferation via a predominant RhoA/ROCK‐dependent mechanism. These results suggest an important role for this integrin in intestinal crypt cell homoeostasis.     

CONTROL OF EPITHELIAL CELL PROLIFERATION IN THE SMALL INTESTINAL CRYPT

A heat labile factor which has been shown to inhibit proliferative activity in crypt epithelium both in rat jejunum in vivo and in explants of rat jejunum maintained in organ culture has been prepared from the soluble fraction of homogenized epithelial cells isolated from rat small intestinal crypts. The factor appears to have tissue specificity, for it has no influence on epithelial cell proliferation in colonic crypts, oesophagus or skin. Extracts of rat intestinal villous cells prepared using identical techniques were without effect on proliferative activity of small intestinal crypt epithelium.
Isoprenalin, which was also found to suppress cell proliferation, did not potentiate the effect of the factor and its effects were evanescent.     

Study of the protective effect on damaged intestinal epithelial cells of rat multilineage‐differentiating stress‐enduring (Muse) cells

In this study, we determined whether multilineage‐differentiating stress‐enduring (Muse) cells exist in rat bone marrow and elucidated their effects on protection against the injury of intestinal epithelial cells associated with inflammation. Rat Muse cells were separated from bone marrow mesenchymal stem cells (BMMSCs) by trypsin‐incubation stress. The group of cells maintained the characteristics of BMMSCs; however, there were high positive expression levels of stage‐specific embryonic antigen‐3 (SSEA‐3; 75.6 ± 2.8%) and stage‐specific embryonic antigen‐1 (SSEA‐1; 74.8 ± 3.1%), as well as specific antigens including Nanog, POU class 5 homeobox 1 (OCT 3/4), and SRY‐box 2 (SOX 2). After inducing differentiation, α‐fetoprotein (endodermal), α‐smooth muscle actin and neurofilament medium polypeptide (ectodermal) were positive in Muse cells. Injuries of intestinal epithelial crypt cell‐6 (IEC‐6) and colorectal adenocarcinoma 2 (Caco‐2) cells as models were induced by tumor necrosis factor‐α stimulation in vitro. Muse cells exhibited significant protective effects on the proliferation and intestinal barrier structure, the underlying mechanisms of which were related to reduced levels of interleukin‐6 (IL‐6) and interferon‐γ (IFN‐γ), and the restoration of transforming growth factor‐β (TGF‐β) and IL‐10 in the inflammation microenvironment. In summary, there were minimal levels of pluripotent stem cells in rat bone marrow, which exhibit similar properties to human Muse cells. Rat Muse cells could provide protection against damage to intestinal epithelial cells depending on their anti‐inflammatory and immune regulatory functionality. Their functional impact was more obvious than that of BMMSCs.     

Scanning electron microscopy of isolated epithelium of the murine gastrointestinal tract: morphology of the basal surface and evidence for paracrinelike cells

By using the method of Bjerknes and Cheng, isolated murine gastrointestinal epithelial sheets were prepared for scanning electron microscopy. Examination of isolated epithelium from fundic stomach revealed numerous branched gastric glands. Parietal cells were easily detected bulging from the basal surface of the glandular epithelium. The basal surface membrane of parietal cells appeared smooth, with only sparse microvilluslike projections, whereas adjacent glandular cells had numerous 1- to 2-micron fingerlike projections which interdigitated laterally with similar processes from adjacent cells. Occasionally, paracrinelike cells having long cytoplasmic processes ranging from 10 to 20 micron in length were observed on the basal epithelial surface of the stomach and the colon, but not the small intestine. In isolated intestinal epithelia, the basal surface of crypt epithelial cells showed extensive cytoplasmic interdigitations, but no distinct morphology permitting recognition of individual cell types. Various stages of intestinal crypt bifurcation were seen. Craterlike spaces in the basal surface of crypt epithelium, presumably due to migrating leukocytes, were also numerous. Examination of the luminal surface of the isolated intestinal epithelium revealed that intimate associations between epithelium and mucosal-associated microorganisms were maintained, thus suggesting that minimal alterations in surface morphology were incurred by epithelial isolation. These observations on epithelial structure suggest that isolated gastrointestinal epithelia may be well suited for physiological studies of epithelial function and interactions with the microbial flora.     

肠道干细胞和潘氏细胞在肠道稳态和疾病中的作用

肠道是最复杂的器官之一,负责营养的吸收和消化。肠道具有多层结构保护整个肠道免受病原体的侵害。肠道上皮是由单层柱状上皮细胞组成,是抵抗病原体的第一道屏障。因此,肠上皮必须保持完整性以保护肠免受感染和毒性剂的侵害。上皮细胞分为两个谱系(吸收型与分泌型),并且每隔3~4天脱落至肠腔中。细胞的快速更替是由于肠道干细胞的存在,肠道干细胞排列在隐窝底部终极分化的潘氏细胞之间并沿隐窝绒毛轴分化成不同的上皮细胞。一旦肠道干细胞受到损伤,潘氏细胞将通过提供WNT配体和Notch刺激来补充肠道干细胞。因此,潘氏细胞充当辅助细胞以维持干细胞微环境,即生态位。该综述探讨了干细胞和潘氏细胞之间的相互作用,进一步探讨了维持肠道稳态的信号通路。     

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.     

Evaluation of the radiation response and regenerative effects of mesenchymal stem cell-conditioned medium in an intestinal organoid system

Intestinal organoids have recently emerged as an in vitro model relevant to the gut system owing to their recapitulation of the native intestinal epithelium with crypt–villus architecture. However, it is unclear whether intestinal organoids reflect the physiology of the in vivo stress response. Here, we systemically investigated the radiation response in organoids and animal models using mesenchymal stem cell-conditioned medium (MSC-CM), which contains secreted paracrine factors. Irradiated organoids exhibited sequential induction of viability loss and regrowth after irradiation (within 12 days), similar to the response of the native intestinal epithelium. Notably, treatment with MSC-CM facilitated the reproliferation of intestinal stem cells (ISCs) and restoration of damaged crypt-villus structures in both models. Furthermore, Wnt/Notch signaling pathways were commonly upregulated by MSC-CM, but not radiation, and pharmacologically selective inhibition of Wnt or Notch signaling attenuated the enhanced recovery of irradiated organoids, with increases in ISCs, following MSC-CM treatment. Interestingly, the expression of Wnt4, Wnt7a, and active β-catenin was increased, but not notch family members, in MSC-CM-treated organoid after irradiation. Treatment of recombinant mouse Wnt4 and Wnt7a after irradiation improved to some extent intestinal epithelial regeneration both in vitro and in vivo. Overall, these results suggested that intestinal organoids recapitulated the physiological stress response of the intestinal epithelium in vivo. Thus, our findings provided important insights into the physiology of intestinal organoids and may contribute to the development of strategies to enhance the functional maturation of engineered organoids.     

STORM: a general model to determine the number and adaptive changes of epithelial stem cells in teleost, murine and human intestinal tracts

Intestinal stem cells play a pivotal role in the epithelial tissue renewal, homeostasis and cancer development. The lack of a general marker for intestinal stem cells across species has hampered analysis of stem cell number in different species and their adaptive changes upon intestinal lesions or during development of cancer. Here a two-dimensional model, named STORM, has been developed to address this issue. By optimizing epithelium renewal dynamics, the model examines the epithelial stem cell number by taking experimental input information regarding epithelium proliferation and differentiation. As the results suggest, there are 2.0-4.1 epithelial stem cells on each pocket section of zebrafish intestine, 2.0-4.1 stem cells on each crypt section of murine small intestine and 1.8-3.5 stem cells on each crypt section of human duodenum. The model is able to provide quick results for stem cell number and its adaptive changes, which is not easy to measure through experiments. Its general applicability to different species makes it a valuable tool for analysis of intestinal stem cells under various pathological conditions.     

Canonical and noncanonical inflammasomes in intestinal epithelial cells

Inflammasomes are cytosolic, multimeric protein complexes capable of activating pro‐inflammatory cytokines such as IL‐1β and IL‐18, which play a key role in host defence. Inflammasome components are highly expressed in the intestinal epithelium. In recent years, studies have begun to demonstrate that epithelial‐intrinsic inflammasomes play a critical role in regulating epithelial homeostasis, both by defending the epithelium from pathogenic insult and through the regulation of the mucosal environment. However, the majority of research regarding inflammasome activation has focused on professional immune cells, such as macrophages. Here, we present an overview of the current understanding of inflammasome function in epithelial cells and at mucosal surfaces and, in particular, in the intestine.     

Rat intestinal epithelial cells modify the reactivity of bovine milk lactoferrin to concanavalin A

Bovine milk lactoferrin suppressed proliferation of concanavalin A-stimulated rat spleen lymphocytes by absorbing mitogenic lectin activity. Culture media, conditioned by incubating allogeneic intestinal epithelial villus and crypt cells with or without lactoferrin, also suppressed the proliferation. Villus cells absorbed lactoferrin during preparation of conditioned medium and the medium lost a lactoferrin-dependent lymphocyte proliferation-suppressing activity. Although crypt cells did not absorb lactoferrin, its conditioned medium lost the activity. These conditioned media did not alter proliferation of lymphocytes stimulated with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin. Serum proteins, albumin and transferrin, did not substitute for milk lactoferrin. Thus, intestinal epithelial cells modified the reactivity of milk lactoferrin to concanavalin A.     

肠道上皮与肠道微生物相互作用的研究现状

肠道上皮是肠上皮细胞及其分泌物有机构成的黏膜界面。随着技术的进步和对肠道菌群作用的逐渐重视,研究者对肠道上皮与肠道微生物相互作用的认识也不断深入。研究表明,肠道上皮调节并维持肠道微生物的定殖与分布,肠道微生物也影响肠道上皮的多种屏障功能,二者通过一系列细胞分子机制紧密联系,共同维持肠道稳态。此外,其过程中产生的宿主-肠道菌群共代谢物被发现可以反映宿主的生理病理状态,作为指标被应用于临床疾病诊断、治疗效果评估和预后推测。本文基于近年的研究,综述了肠道上皮与肠道微生物的相互作用及其细胞分子机制,为进一步研究和临床应用总结了理论基础,并探讨了未来可能的研究方向。     

Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR‐8–Escargot switch

The intestinal epithelium is remarkably robust despite perturbations and demand uncertainty. Here, we investigate the basis of such robustness using novel tracing methods that allow simultaneously capturing the dynamics of stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. We found that intestinal stem cells (ISCs) divide “ahead” of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell–cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial–mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR‐8/miR‐200 in “pausing” enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse MET by antagonizing escargot. Our findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand.     

The protective potency of probiotic bacteria and their microbial products against enteric infections-review

The intestinal environment accommodates a wide range of contents ranging from harmless beneficial dietary and microbial flora to harmful pathogenic bacteria. This has resulted in the development of highly adapted epithelial cells lining the intestine. This adaptation involves the potential of crypt cells to proliferate and to constantly replace villous cells that are lost due to maturity or death. As a result, the normal intestinal epithelial integrity and functions are maintained. This phenomenon is eminent in intestinal defense whereby the intestinal epithelial cells serve as a physical barrier against luminal agents. The protection against agents in the gut lumen can only be effective if the epithelium is intact. Restitution of the damaged epithelium is therefore crucial in this type of defense.