技术资料

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore,one stem cell might easily account for the renewal of mammary epithelium over several transplant generations. View Publication

An in vivo transplantation system has been used to evaluate the developmental capacities of specific mouse mammary epithelial cell populations. Specifically,mouse mammary epithelial cells with distinctly limited developmental potentials have been identified using this procedure. Two distinct epithelial cell progenitors have been identified by experiments designed to determine whether basal lobular and ductal phenotypes could develop independently under conditions imposed by a limiting dilution. The prediction that these separate epithelial progenitors must exist was based upon the results from transplantation experiments carried out in epithelium-divested mammary fat pads of syngeneic mice with mammary epithelium from two different transgenic mouse models. The results presented here demonstrate the following points: 1) lobular,i.e. secretory,progenitor cells are present as distinct entities among the mammary epithelial cells found in immature virgin female mice; 2) similarly,ductal epithelial progenitors are present within the same population; 3) lobular progenitors are present in greater numbers,although both cell populations are extremely small; 4) as expected,some inocula produce outgrowths with simultaneous development of both lobular and ductal phenotypes--it is not known whether this indicates cooperative interaction between the two epithelial progenitors or signals the presence of a third progenitor type capable of producing both ductular and lobular committed daughters; 5) these findings have important consequences in the design of experiments aimed at testing the effects of known and putative mammary oncogenes and tumor suppressor genes,using techniques which include cellular transformation in vitro followed by in vivo cultivation and evaluation. View Publication

Total-body exposure to radiation causes widespread tissue injury. Damage to the hematopoietic and intestinal stem cell compartments is particularly lethal and mitigators of this damage are critical in providing effective treatment. Parabiosis radiation experiments,in which the vasculatures of two rodents are anastomosed prior to irradiation of one of the animals,have shown that there is a circulating factor that protects mice from radiation-induced intestinal death. Recently reported studies have suggested that growth differentiation factor 11 (GDF11) is responsible for the rejuvenation of stem cells observed in parabiosis experiments involving aging mice. In this study,we investigated the efficacy of GDF11 as a potential mitigator of radiation-induced damage to intestinal stem cells. In ex vivo cultures of intestinal organoids,the number of cells expressing the stem cell marker Lgr5 was increased after irradiation and GDF11 supplementation. Further ex vivo studies to assess stem cell function,measured by the ability to grow new crypt-like structures,did not show increased stem cell activity in response to GDF11 treatment. In addition,GDF11 was unable to improve survival of mice subjected to total-abdominal irradiation. These data demonstrate that GDF11 does not mitigate radiation damage to intestinal stem cells. View Publication

Intestinal tuft cells are one of 4 secretory cell linages in the small intestine and the source of IL-25,a critical initiator of the type 2 immune response to parasite infection. When Raptor,a critical scaffold protein for mammalian target of rapamycin complex 1 (mTORC1),was acutely deleted in intestinal epithelium via Tamoxifen injection in Tritrichomonas muris (Tm) infected mice,tuft cells,IL-25 in epithelium and IL-13 in the mesenchyme were significantly reduced,but Tm burden was not affected. When Tm infected mice were treated with rapamycin,DCLK1 and IL-25 expression in enterocytes and IL-13 expression in mesenchyme were diminished. After massive small bowel resection,tuft cells and Tm were diminished due to the diet used postoperatively. The elimination of Tm and subsequent re-infection of mice with Tm led to type 2 immune response only in WT,but Tm colonization in both WT and Raptor deficient mice. When intestinal organoids were stimulated with IL-4,tuft cells and IL-25 were induced in both WT and Raptor deficient organoids. In summary,our study reveals that enterocyte specific Raptor is required for initiating a type 2 immune response which appears to function through the regulation of mTORC1 activity. View Publication

Type I and type III interferons (IFNs) are crucial components of the first-line antiviral host response. While specific receptors for both IFN types exist,intracellular signaling shares the same Jak-STAT pathway. Due to its receptor expression,IFN-λ responsiveness is restricted mainly to epithelial cells. Here,we display IFN-stimulated gene induction at the single cell level to comparatively analyze the activities of both IFN types in intestinal epithelial cells and mini-gut organoids. Initially,we noticed that the response to both types of IFNs at low concentrations is based on a single cell decision-making determining the total cell intrinsic antiviral activity. We identified histone deacetylase (HDAC) activity as a crucial restriction factor controlling the cell frequency of IFN-stimulated gene (ISG) induction upon IFN-λ but not IFN-β stimulation. Consistently,HDAC blockade confers antiviral activity to an elsewise non-responding subpopulation. Second,in contrast to the type I IFN system,polarization of intestinal epithelial cells strongly enhances their ability to respond to IFN-λ signaling and raises the kinetics of gene induction. Finally,we show that ISG induction in mini-gut organoids by low amounts of IFN is characterized by a scattered heterogeneous responsiveness of the epithelial cells and HDAC activity fine-tunes exclusively IFN-λ activity. This study provides a comprehensive description of the differential response to type I and type III IFNs and demonstrates that cell polarization in gut epithelial cells specifically increases IFN-λ activity. View Publication

Sterol regulatory element-binding protein-2 (SREBP-2) activates transcription of all genes needed for cholesterol biosynthesis. To study SREBP-2 function in intestine,we generated a mouse model (Vil-BP2(-/-) ) in which Cre recombinase ablates SREBP-2 in intestinal epithelia. Intestines of Vil-BP2(-/-) mice had reduced expression of genes required for sterol synthesis,in vivo sterol synthesis rates,and epithelial cholesterol contents. On a cholesterol-free diet,they displayed chronic enteropathy with histological abnormalities of both villi and crypts,growth restriction,and reduced survival that was prevented by supplementation of cholesterol in the diet. Likewise,SREBP-2-deficient enteroids required exogenous cholesterol for growth. Blockade of luminal cholesterol uptake into enterocytes with ezetimibe precipitated acutely lethal intestinal damage in Vil-BP2(-/-) mice,highlighting the critical interplay in the small intestine of sterol absorption via NPC1L1 and sterol synthesis via SREBP-2 in sustaining the intestinal mucosa. These data show that small intestine requires SREBP-2 to drive cholesterol synthesis that sustains the intestinal epithelia when uptake of cholesterol from the gut lumen is not available,and provide a unique example of cholesterol auxotrophy expressed in an intact,adult mammal. View Publication

The current in vitro or in vivo intestinal fibrosis models have many limitations. Recent advancements in the isolation and culturing of organoids has led to development of various three-dimensional (3D) intestinal disease models with in vivo physiology. In this study,we generated an organoid-based epithelial to mesenchymal transition (OEMT) model,which could be used as a novel intestinal fibrosis model. Intestinal epithelial organoids (IEOs) were isolated and cultured from the small intestines of normal mice. IEOs were treated with transforming growth factor- β1 (TGF-β1) or Tumor necrosis factor-α (TNF-α) to evaluate their phenotypic change. Raw 264.7 cells (macrophage) stimulated with lipopolysaccharide were co-cultured with IEOs in growth media with or without TGF-β1. TGF-β1 alone slightly induced epithelial to mesenchymal transition (EMT) in the IEOs but mainly disrupted them. Macrophage released cytokines synergistically induced mesenchymal phenotypic changes in TGF-β1 stimulated intestinal organoids. TNF-α and TGF-β1 synergistically induced proliferation of mesenchymal cells as well as EMT in the IEOs. We generated a novel OEMT model based on our finding that TNF-α and TGF-β synergistically induce type 2 EMT in IEOs. This 3D EMT model with in vivo physiology could be used to study EMT associated intestinal fibrosis. View Publication

Irregular mitochondria structure and reduced ATP in some patients with IBD suggest that metabolic stress contributes to disease. Loss-of-function mutation in the nucleotide-binding oligomerization domain (NOD)-2 gene is a major susceptibility trait for IBD. Hence,we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with noninvasive Escherichia coli + DNP (16 h). Increased intracellular bacteria in wild-type (WT) and NOD2 knockdown (KD) cells and colonoids from NOD2(-/-) mice were mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by cotreatment with the antioxidant mitoTEMPO and the ERK inhibitor U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared with WT cells,there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus,in a two-hit hypothesis,decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently,greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations have compromised macrophage and Paneth cell responses to bacteria.NEW & NOTEWORTHY Increased internalization of bacteria by epithelia with dysfunctional mitochondria (reduced ATP) is potentiated if the cells lack nucleotide-binding oligomerization domain 2 (NOD2),mutations in which are inflammatory bowel disease-susceptibility traits. Uptake of bacteria was dependent on reactive oxygen species and MAP-kinase activity,and the increased viable intracellular bacteria in NOD2(-/-) cells likely reflect a reduced ability to recognize and kill bacteria. Thus a significant barrier defect occurs with NOD2 deficiency in conjunction with metabolic stress that could contribute to inflammation. View Publication

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure,the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches,the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling,and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice,the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked,demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens,inflammatory stimuli and aging can modify M cell-density in the gut,these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice,resulting in shortened survival times and increased disease susceptibility,equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection,whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases. View Publication

Incorporation of thymidine analogues in replicating DNA,coupled with antibody and fluorophore staining,allows analysis of cell proliferation,but is currently limited to monolayer cultures,fixed cells and end-point assays. We describe a simple microscopy imaging method for live real-time analysis of cell proliferation,S phase progression over several division cycles,effects of anti-proliferative drugs and other applications. It is based on the prominent (˜ 1.7-fold) quenching of fluorescence lifetime of a common cell-permeable nuclear stain,Hoechst 33342 upon the incorporation of 5-bromo-2'-deoxyuridine (BrdU) in genomic DNA and detection by fluorescence lifetime imaging microscopy (FLIM). We show that quantitative and accurate FLIM technique allows high-content,multi-parametric dynamic analyses,far superior to the intensity-based imaging. We demonstrate its uses with monolayer cell cultures,complex 3D tissue models of tumor cell spheroids and intestinal organoids,and in physiological study with metformin treatment. View Publication