Neurosphere cultures treated with different doses of LPS

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Compromised Tissue Renewal in the Ageing Human Colonic EpitheliumEsther M. Mitchell, Alyson Parris, Loren Bigwood, Natalia Scobioala-laker, Amy Reynolds,Michael P. Lewis, Wing Leung, Nigel Belshaw, Ian T. Johnson, Naohide Oue, WataruYasui, Ian Beales, Crawford P. Jamieson, Mark Tremelling, Richard Tighe, Alison Prior,Mark Williams

BACKGROUND: The intestinal epithelium is the most rapidly renewing tissue in the body.It is widely believed that this attribute minimises the accumulation of age-related moleculardamage. Increasing evidence suggests that this protective mechanism is undermined by age-related molecular changes that accumulate in long-lived stem/progenitor cells. Age-relatedmolecular damage in the intestinal epithelium of flies and rodents manifests as a hyperprolifer-ative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reducedregenerative potential following damage. Furthermore, the human colonic epithelium issubject to age-related accumulation of mutations in mitochondrial DNA. Given that ageingis a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageinghuman colonic epithelium has received little attention. AIM: To investigate age-relatedchanges in the renewal of the human colonic epithelium.METHODS: Tissue biopsies obtainedat sigmoidoscopy (Ethical approval) from young (<40 years, N=11) and old (>70 years, N=12) individuals with no apparent pathology were immediately fixed or processed for cryptisolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy.Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morpho-metric analysis and immunofluorescence for detection of Ki67 (proliferation marker), betacatenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cellmarker). RESULTS: Crypts from young subjects (n=116 crypts) were significantly longer(p< 0.05) than those derived from the older cohort (117 crypts), 375± 10 um versus 330± 10 um, respectively. The percentage of Ki67 positive cells in all regions along the crypt-axis was significantly greater (p<0.05) in tissue from older subjects : e.g. crypt base - 35%(young) versus 52% (old); mid region - 42%% (young) versus 62% (old) and upper region10% (young) versus 17% (old). The number of cell divisions observed under timelapsemicroscopy was reduced for crypts derived from older subjects suggesting that the increasedKi67 labelling index in the older group reflected a slower cell cycle time. Although thenature of the proliferating cell type is not known at this stage, an increase in OLFM4+(stem) cell number was observed along the axis of crypts derived from older subjects. Theabove traits were associated with a trend towards an extended profile of immunolabellingintensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis oftissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length,cell proliferation and markers for intestinal stem cells and Wnt signalling componentsalong the crypt-axis suggest that tissue renewal is compromised in the ageing humancolonic epithelium.

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Defining the Function of the Putative Stem Cell Marker Musashi1 in IntestinalPhysiopathologyFrancesca Cambuli, Michela Plateroti

Introduction. Musashi1 is a known marker of somatic stem cells in various tissues includingthe intestinal epithelium. Musashi1 is a RNA-binding protein involved in stemness in Droso-phila and in mammals (rev. in Okano et al., 2005). It has also been shown that its expressionis upregulated in intestinal tumours from Apc-mutant mice (Potten et al., 2003). Data alsoshowed that Musashi1 induces cell proliferation (Wang et al., 2008) and growth of tumorcells (Sureban et al., 2008). Our recent study in intestinal epithelium primary culturesshowed that Musashi1 overexpression promotes progenitors proliferation and activates Wntand Notch pathways. Moreover, Msi1-overexpressing cells display tumorigenic propertiesin xenograft experiments. (Rezza et al., 2010). We sought to analyze here the function ofMusashi1 in the intestinal physiopathology in the mouse In Vivo. Methods. We generatedthe transgenic mice v-Mus that express Musashi1 cDNA under the control of the murineVillin promoter. This leads to Musashi1 expression all along the crypt-villus/crypt-surfaceaxis, starting early during embryo development. Results. First, we confirmed the expressionof the transgene specifically in the epithelium of the small and large intestine of the v-Musmice, compared with the WT littermates. Then we verified that the expression of Musashi1is significantly increased in the v-Mus compared with the WT animals, both at mRNA andprotein level as analyzed by RTqPCR and WB. Finally, by immunohistochemical approachwe showed that Musashi1 is expressed by all the epithelial cells. On the contrary, as previouslydescribed (Potten et al., 2003), in WT mice only a few cells per crypt express Musashi1.The intestine of v-Mus and WT mice has been collected from one and three month-oldanimals and we started the analysis of the intestinal phenotype. Interestingly, in v-Musanimals we could observe an extension of the proliferative units in both the small and largeintestine at both ages. We are currently analyzing, by specific immunostaining, the differentcytotypes composing the intestinal epithelium to define whether the expansion of the progen-itor zone, is associated with decreased differentiation capacities. Conclusions and perspect-ives. The over- and ectopic expression of Musashi1 induces in animals In Vivo an expansionof the proliferative compartment. Studies in aged animals will enable us to define whetherit can have oncogenic properties In Vivo, as we previously demonstrated in xenograft experi-ments. In conclusion, these mice are a powerful tool to study the function of Musashi1 inthe intestine In Vivo. They will also enable us to analyze in detail the mechanisms of Musashi1-associated oncogenic properties in the process of intestinal tumorigenesis.

S-321 AGA Abstracts

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Oct4 Directs TGF-Beta Signaling in Embryonic Stem CellsAlan C. Mullen, Jamie J. Newman, David A. Orlando, Richard A. Young

Aims: TGF-beta signaling plays a critical role in maintaining human embryonic stem (ES)state and directing endodermal differentiation through activation of the transcription factorsSmad2 and Smad3 (Smad2/3). The mechanism by which Smad2/3 are targeted to DNAremains unclear. We found that Smad2/3 tend to co-occupy DNA with the master transcrip-tion factor Oct4 and sought to understand the role of Oct4 in determining the gene targetsof the TGF-beta signaling pathway. We first asked if Oct4 is required for normal Smad3binding in ES cells and then how Oct4 recruits Smad2/3 to gene targets. Methods: We usedchromatin immunoprecipitation (ChIP) to determine whether Smad3 could occupy DNAsites in the absence of Oct4. We next used co-immunoprecipitation (co-IP) to determine ifSmad3 and Oct4 could form a physical complex. We used ChIP followed by massive parallelDNA sequencing (ChIP-seq) to analyze nucleosome occupancy at sites bound by Smad3 andOct4 as well as computational approaches to scan the genome to determine the distribution ofSmad binding elements relative to sites bound by Oct4. Results: Oct4 and Smad3 physicallyinteract, and loss of Oct4 expression results in a loss of Smad3 binding at sites normallyco-occupied by Smad3 and Oct4. Smad binding elements are enriched near sites bound byOct4, and these sites are depleted of nucleosomes. Conclusion: Oct4 is required for Smad2/3 to bind genomic targets in ES cells. Binding of Oct4 repositions nucleosomes to makeadjacent Smad binding elements accessible to Smad2/3, and Oct4 interacts with Smad2/3to stabilize binding to DNA. In this manner, TGF-beta signaling is tailored to regulate thegenes most relevant to ES cells.

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Cutting-Edge: A New Method to Expand Neural Stem Cells of the MyentericPlexus From Adult Mouse by Maintaining the Stem Cell Niche In VitroDavid Grundmann, Franziska Markwart, Karl-Herbert Schäfer

The enteric nervous system (ENS) is subdivided in the Myenteric Plexus (MP) and theSubmucosus Plexus (SP) which participates in intestinal regulation during health and disease.The ENS plays a key role in regulating gastrointestinal tract motility, abdominal pain andcan also be involved in inflammation. The plasticity of the ENS is retained by residing neuralstem cells which possess the capability to expand and to differentiate into neuronal andglial lineages. The interaction of the stem cells with their particular local tissue microenviron-ment, also known as “stem cell niche”, is characterized by cell-cell contacts, autocrine andparacrine as well as endocrine signals, and metabolic products of tissue activity. We thereforeisolated the MP with a high purity from the of adult mouse gut. Tissue was cultured eitherdissociated or undissociated to investigate the influence of the intact plexus microenviron-ment, maintaining the In Vivo stem cell niche In Vitro. The small intestine from adult mousewas used to strip the muscular layer from the submucosar layer, followed by enzymaticaldigestion with purified collagenase (Liberase) and a final mechanical disruption step. Wecultured the harvested MP to expand the neural stem cells and measured the proliferationby the detection and enumeration of generated neurospheres. In addition, we analyzed thecapacity of the In Vitro proliferated stem cells to differentiate into neuronal and glial lineagesby immunofluorescence staining. The underlying mechanisms of the stem cell self-renewalwere analysed by revealing the surrounding cells in the stem cell niche in situ and detectingsoluble auto and paracrine factors by multiplexe assay. In general, leaving the stem cellniche at least partially intact led to a higher yield of neurospheres. Our results demonstratethat the way of using the MP as a stem cell source for therapeutic strategies to cure intestinaldysganglionosis is significantly dependent on the culturing techniques. Optimizing the modeand condition of culture might lead to a sufficient amount of neural stem cells, sufficientfor successful transplantations. Moreover, the isolation of large amounts of pure MP in healthand diseases allows the promising application of proteomic techniques for a much moredetailed analysis.

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Organ-Specific Migration of CD133+ Stem Cells Triggered by CpG MotifsFrom DSS-Induced Colitis Mouse ModelGabor Valcz, Tibor Krenács, Ferenc Sipos, Sándor Spisák, Kinga Tóth, Árpád V. Patai,Alexandra Kalmar, Barnabas Wichmann, Katalin Leiszter, Zsolt Tulassay, Béla Molnár

Background: Following tissue damage, the invasion of inflammatory cells and regenerativestem cells is influenced by chemical regulators, e.g. cytokines and chemokines. Migrating,regenerative stem cells are enriched in Toll-like receptors, which recognize CpG motifs ofbacterial or viral DNA. Tissue damage is well characterized by increased levels of cell freeDNA (cfDNA). Here we hypothesized, that CpG motifs of DNA from DSS-induced mousemay cause an increased organ-specific migration of CD133+ stem cells in healthy mice.Aims: To determine CD133 mRNA expression in the plasma and the alteration of colorectalintraepithelial CD133 protein positive cell number in healthy mice after the injection ofDNA isolated from DSS-treated mouse plasma. Materials and methods: Mouse colitis modelwas established by treatment with 1% DSS, then cfDNA was isolated from DSS-treatedanimals and was injected into healthy mice. Peripherial blood samples were taken 1, 2, 4and 24 hours after the treatment and themRNA expression of CD133, TLR9 and inflammatorycascademembers (such as IL-6, TNF-α) was measured by RT-PCR, where beta-2-microglobu-lin (B2M) was used as reference gene. From formalin fixed and paraffin embedded tissuesamples (colon, spleen, kidney, lung and skin) of the treated animals 4 μm thick sectionswere cut and immunostained for CD133 polyclonal antibody (Abcam, Cambridge, UK).Then the slides were digitalized and analyzed by digital microscopy. Results: After 2 hoursof the DNA injection the expression of all analyzedmarkers increased significantly in periferialblood compared to the normal state. In colonic tissue we found significantly (p<0,01)increased number of intraepithelial CD133+ cells in the DNA treated healthy mice(9,86±0,84%) compared to the control (7,36±1,1%) animals. Histologically the colonicepithelium showed increased number of goblet cells, as a sign of elevated regeneration. Incontrast, no considerable morphological alteration or stem cell influx could be observed in

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