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Defining the Function of the Putative Stem Cell Marker Musashi1 in Intestinal Physiopathology
Sa1758
Aims: TGF-beta signaling plays a critical role in maintaining human embryonic stem (ES) state and directing endodermal differentiation through activation of the transcription factors Smad2 and Smad3 (Smad2/3). The mechanism by which Smad2/3 are targeted to DNA remains unclear. We found that Smad2/3 tend to co-occupy DNA with the master transcription factor Oct4 and sought to understand the role of Oct4 in determining the gene targets of the TGF-beta signaling pathway. We first asked if Oct4 is required for normal Smad3 binding in ES cells and then how Oct4 recruits Smad2/3 to gene targets. Methods: We used chromatin immunoprecipitation (ChIP) to determine whether Smad3 could occupy DNA sites in the absence of Oct4. We next used co-immunoprecipitation (co-IP) to determine if Smad3 and Oct4 could form a physical complex. We used ChIP followed by massive parallel DNA sequencing (ChIP-seq) to analyze nucleosome occupancy at sites bound by Smad3 and Oct4 as well as computational approaches to scan the genome to determine the distribution of Smad binding elements relative to sites bound by Oct4. Results: Oct4 and Smad3 physically interact, and loss of Oct4 expression results in a loss of Smad3 binding at sites normally co-occupied by Smad3 and Oct4. Smad binding elements are enriched near sites bound by Oct4, 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 make adjacent Smad binding elements accessible to Smad2/3, and Oct4 interacts with Smad2/3 to stabilize binding to DNA. In this manner, TGF-beta signaling is tailored to regulate the genes most relevant to ES cells.
Neurosphere cultures treated with different doses of LPS Sa1756 Compromised Tissue Renewal in the Ageing Human Colonic Epithelium Esther M. Mitchell, Alyson Parris, Loren Bigwood, Natalia Scobioala-laker, Amy Reynolds, Michael P. Lewis, Wing Leung, Nigel Belshaw, Ian T. Johnson, Naohide Oue, Wataru Yasui, 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 molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium. METHODS: Tissue biopsies obtained at 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 crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). 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 cryptaxis 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 region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature 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. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.
Sa1759 Cutting-Edge: A New Method to Expand Neural Stem Cells of the Myenteric Plexus From Adult Mouse by Maintaining the Stem Cell Niche In Vitro David Grundmann, Franziska Markwart, Karl-Herbert Schäfer The enteric nervous system (ENS) is subdivided in the Myenteric Plexus (MP) and the Submucosus Plexus (SP) which participates in intestinal regulation during health and disease. The ENS plays a key role in regulating gastrointestinal tract motility, abdominal pain and can also be involved in inflammation. The plasticity of the ENS is retained by residing neural stem cells which possess the capability to expand and to differentiate into neuronal and glial lineages. The interaction of the stem cells with their particular local tissue microenvironment, also known as “stem cell niche”, is characterized by cell-cell contacts, autocrine and paracrine as well as endocrine signals, and metabolic products of tissue activity. We therefore isolated the MP with a high purity from the of adult mouse gut. Tissue was cultured either dissociated or undissociated to investigate the influence of the intact plexus microenvironment, maintaining the In Vivo stem cell niche In Vitro. The small intestine from adult mouse was used to strip the muscular layer from the submucosar layer, followed by enzymatical digestion with purified collagenase (Liberase) and a final mechanical disruption step. We cultured the harvested MP to expand the neural stem cells and measured the proliferation by the detection and enumeration of generated neurospheres. In addition, we analyzed the capacity of the In Vitro proliferated stem cells to differentiate into neuronal and glial lineages by immunofluorescence staining. The underlying mechanisms of the stem cell self-renewal were analysed by revealing the surrounding cells in the stem cell niche in situ and detecting soluble auto and paracrine factors by multiplexe assay. In general, leaving the stem cell niche at least partially intact led to a higher yield of neurospheres. Our results demonstrate that the way of using the MP as a stem cell source for therapeutic strategies to cure intestinal dysganglionosis is significantly dependent on the culturing techniques. Optimizing the mode and condition of culture might lead to a sufficient amount of neural stem cells, sufficient for successful transplantations. Moreover, the isolation of large amounts of pure MP in health and diseases allows the promising application of proteomic techniques for a much more detailed analysis.
Sa1757 Defining the Function of the Putative Stem Cell Marker Musashi1 in Intestinal Physiopathology Francesca Cambuli, Michela Plateroti Introduction. Musashi1 is a known marker of somatic stem cells in various tissues including the intestinal epithelium. Musashi1 is a RNA-binding protein involved in stemness in Drosophila and in mammals (rev. in Okano et al., 2005). It has also been shown that its expression is upregulated in intestinal tumours from Apc-mutant mice (Potten et al., 2003). Data also showed that Musashi1 induces cell proliferation (Wang et al., 2008) and growth of tumor cells (Sureban et al., 2008). Our recent study in intestinal epithelium primary cultures showed that Musashi1 overexpression promotes progenitors proliferation and activates Wnt and Notch pathways. Moreover, Msi1-overexpressing cells display tumorigenic properties in xenograft experiments. (Rezza et al., 2010). We sought to analyze here the function of Musashi1 in the intestinal physiopathology in the mouse In Vivo. Methods. We generated the transgenic mice v-Mus that express Musashi1 cDNA under the control of the murine Villin promoter. This leads to Musashi1 expression all along the crypt-villus/crypt-surface axis, starting early during embryo development. Results. First, we confirmed the expression of the transgene specifically in the epithelium of the small and large intestine of the v-Mus mice, compared with the WT littermates. Then we verified that the expression of Musashi1 is significantly increased in the v-Mus compared with the WT animals, both at mRNA and protein level as analyzed by RTqPCR and WB. Finally, by immunohistochemical approach we showed that Musashi1 is expressed by all the epithelial cells. On the contrary, as previously described (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-old animals and we started the analysis of the intestinal phenotype. Interestingly, in v-Mus animals we could observe an extension of the proliferative units in both the small and large intestine at both ages. We are currently analyzing, by specific immunostaining, the different cytotypes composing the intestinal epithelium to define whether the expansion of the progenitor zone, is associated with decreased differentiation capacities. Conclusions and perspectives. The over- and ectopic expression of Musashi1 induces in animals In Vivo an expansion of the proliferative compartment. Studies in aged animals will enable us to define whether it can have oncogenic properties In Vivo, as we previously demonstrated in xenograft experiments. In conclusion, these mice are a powerful tool to study the function of Musashi1 in the intestine In Vivo. They will also enable us to analyze in detail the mechanisms of Musashi1associated oncogenic properties in the process of intestinal tumorigenesis.
Sa1760 Organ-Specific Migration of CD133+ Stem Cells Triggered by CpG Motifs From DSS-Induced Colitis Mouse Model Gabor 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 regenerative stem 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 of bacterial or viral DNA. Tissue damage is well characterized by increased levels of cell free DNA (cfDNA). Here we hypothesized, that CpG motifs of DNA from DSS-induced mouse may 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 colorectal intraepithelial CD133 protein positive cell number in healthy mice after the injection of DNA isolated from DSS-treated mouse plasma. Materials and methods: Mouse colitis model was established by treatment with 1% DSS, then cfDNA was isolated from DSS-treated animals and was injected into healthy mice. Peripherial blood samples were taken 1, 2, 4 and 24 hours after the treatment and the mRNA expression of CD133, TLR9 and inflammatory cascade members (such as IL-6, TNF-α) was measured by RT-PCR, where beta-2-microglobulin (B2M) was used as reference gene. From formalin fixed and paraffin embedded tissue samples (colon, spleen, kidney, lung and skin) of the treated animals 4 μm thick sections were cut and immunostained for CD133 polyclonal antibody (Abcam, Cambridge, UK). Then the slides were digitalized and analyzed by digital microscopy. Results: After 2 hours of the DNA injection the expression of all analyzed markers increased significantly in periferial blood 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 colonic epithelium showed increased number of goblet cells, as a sign of elevated regeneration. In contrast, no considerable morphological alteration or stem cell influx could be observed in
S-321
AGA Abstracts
AGA Abstracts
Oct4 Directs TGF-Beta Signaling in Embryonic Stem Cells Alan 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 factors Smad2 and Smad3 (Smad2/3). The mechanism by which Smad2/3 are targeted to DNA remains unclear. We found that Smad2/3 tend to co-occupy DNA with the master transcription factor Oct4 and sought to understand the role of Oct4 in determining the gene targets of the TGF-beta signaling pathway. We first asked if Oct4 is required for normal Smad3 binding in ES cells and then how Oct4 recruits Smad2/3 to gene targets. Methods: We used chromatin immunoprecipitation (ChIP) to determine whether Smad3 could occupy DNA sites in the absence of Oct4. We next used co-immunoprecipitation (co-IP) to determine if Smad3 and Oct4 could form a physical complex. We used ChIP followed by massive parallel DNA sequencing (ChIP-seq) to analyze nucleosome occupancy at sites bound by Smad3 and Oct4 as well as computational approaches to scan the genome to determine the distribution of Smad binding elements relative to sites bound by Oct4. Results: Oct4 and Smad3 physically interact, and loss of Oct4 expression results in a loss of Smad3 binding at sites normally co-occupied by Smad3 and Oct4. Smad binding elements are enriched near sites bound by Oct4, 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 make adjacent Smad binding elements accessible to Smad2/3, and Oct4 interacts with Smad2/3 to stabilize binding to DNA. In this manner, TGF-beta signaling is tailored to regulate the genes most relevant to ES cells.
Neurosphere cultures treated with different doses of LPS Sa1756 Compromised Tissue Renewal in the Ageing Human Colonic Epithelium Esther M. Mitchell, Alyson Parris, Loren Bigwood, Natalia Scobioala-laker, Amy Reynolds, Michael P. Lewis, Wing Leung, Nigel Belshaw, Ian T. Johnson, Naohide Oue, Wataru Yasui, 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 molecular damage. Increasing evidence suggests that this protective mechanism is undermined by agerelated molecular changes that accumulate in long-lived stem/progenitor cells. Age-related molecular damage in the intestinal epithelium of flies and rodents manifests as a hyperproliferative state that exhibits a greater degree of apoptosis, clonogen/stem cell number and reduced regenerative potential following damage. Furthermore, the human colonic epithelium is subject to age-related accumulation of mutations in mitochondrial DNA. Given that ageing is a major risk factor for cancer, it is surprising that the status of tissue renewal in the ageing human colonic epithelium has received little attention. AIM: To investigate age-related changes in the renewal of the human colonic epithelium. METHODS: Tissue biopsies obtained at 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 crypt isolation. Isolated crypts were observed in 3D culture by digital time-lapse microscopy. Native crypts obtained by microdissection of fixed biopsy tissue were subjected to morphometric analysis and immunofluorescence for detection of Ki67 (proliferation marker), beta catenin, c-Myc and axin2 (all markers for Wnt signals), and OLFM4 (an intestinal stem cell marker). 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 cryptaxis 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 region 10% (young) versus 17% (old). The number of cell divisions observed under timelapse microscopy was reduced for crypts derived from older subjects suggesting that the increased Ki67 labelling index in the older group reflected a slower cell cycle time. Although the nature 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. The above traits were associated with a trend towards an extended profile of immunolabelling intensity for nuclear beta catenin and Wnt target gene expression along the crypt-axis of tissue derived from older subjects. CONCLUSIONS: Age-related changes in crypt length, cell proliferation and markers for intestinal stem cells and Wnt signalling components along the crypt-axis suggest that tissue renewal is compromised in the ageing human colonic epithelium.
Sa1759 Cutting-Edge: A New Method to Expand Neural Stem Cells of the Myenteric Plexus From Adult Mouse by Maintaining the Stem Cell Niche In Vitro David Grundmann, Franziska Markwart, Karl-Herbert Schäfer The enteric nervous system (ENS) is subdivided in the Myenteric Plexus (MP) and the Submucosus Plexus (SP) which participates in intestinal regulation during health and disease. The ENS plays a key role in regulating gastrointestinal tract motility, abdominal pain and can also be involved in inflammation. The plasticity of the ENS is retained by residing neural stem cells which possess the capability to expand and to differentiate into neuronal and glial lineages. The interaction of the stem cells with their particular local tissue microenvironment, also known as “stem cell niche”, is characterized by cell-cell contacts, autocrine and paracrine as well as endocrine signals, and metabolic products of tissue activity. We therefore isolated the MP with a high purity from the of adult mouse gut. Tissue was cultured either dissociated or undissociated to investigate the influence of the intact plexus microenvironment, maintaining the In Vivo stem cell niche In Vitro. The small intestine from adult mouse was used to strip the muscular layer from the submucosar layer, followed by enzymatical digestion with purified collagenase (Liberase) and a final mechanical disruption step. We cultured the harvested MP to expand the neural stem cells and measured the proliferation by the detection and enumeration of generated neurospheres. In addition, we analyzed the capacity of the In Vitro proliferated stem cells to differentiate into neuronal and glial lineages by immunofluorescence staining. The underlying mechanisms of the stem cell self-renewal were analysed by revealing the surrounding cells in the stem cell niche in situ and detecting soluble auto and paracrine factors by multiplexe assay. In general, leaving the stem cell niche at least partially intact led to a higher yield of neurospheres. Our results demonstrate that the way of using the MP as a stem cell source for therapeutic strategies to cure intestinal dysganglionosis is significantly dependent on the culturing techniques. Optimizing the mode and condition of culture might lead to a sufficient amount of neural stem cells, sufficient for successful transplantations. Moreover, the isolation of large amounts of pure MP in health and diseases allows the promising application of proteomic techniques for a much more detailed analysis.
Sa1757 Defining the Function of the Putative Stem Cell Marker Musashi1 in Intestinal Physiopathology Francesca Cambuli, Michela Plateroti Introduction. Musashi1 is a known marker of somatic stem cells in various tissues including the intestinal epithelium. Musashi1 is a RNA-binding protein involved in stemness in Drosophila and in mammals (rev. in Okano et al., 2005). It has also been shown that its expression is upregulated in intestinal tumours from Apc-mutant mice (Potten et al., 2003). Data also showed that Musashi1 induces cell proliferation (Wang et al., 2008) and growth of tumor cells (Sureban et al., 2008). Our recent study in intestinal epithelium primary cultures showed that Musashi1 overexpression promotes progenitors proliferation and activates Wnt and Notch pathways. Moreover, Msi1-overexpressing cells display tumorigenic properties in xenograft experiments. (Rezza et al., 2010). We sought to analyze here the function of Musashi1 in the intestinal physiopathology in the mouse In Vivo. Methods. We generated the transgenic mice v-Mus that express Musashi1 cDNA under the control of the murine Villin promoter. This leads to Musashi1 expression all along the crypt-villus/crypt-surface axis, starting early during embryo development. Results. First, we confirmed the expression of the transgene specifically in the epithelium of the small and large intestine of the v-Mus mice, compared with the WT littermates. Then we verified that the expression of Musashi1 is significantly increased in the v-Mus compared with the WT animals, both at mRNA and protein level as analyzed by RTqPCR and WB. Finally, by immunohistochemical approach we showed that Musashi1 is expressed by all the epithelial cells. On the contrary, as previously described (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-old animals and we started the analysis of the intestinal phenotype. Interestingly, in v-Mus animals we could observe an extension of the proliferative units in both the small and large intestine at both ages. We are currently analyzing, by specific immunostaining, the different cytotypes composing the intestinal epithelium to define whether the expansion of the progenitor zone, is associated with decreased differentiation capacities. Conclusions and perspectives. The over- and ectopic expression of Musashi1 induces in animals In Vivo an expansion of the proliferative compartment. Studies in aged animals will enable us to define whether it can have oncogenic properties In Vivo, as we previously demonstrated in xenograft experiments. In conclusion, these mice are a powerful tool to study the function of Musashi1 in the intestine In Vivo. They will also enable us to analyze in detail the mechanisms of Musashi1associated oncogenic properties in the process of intestinal tumorigenesis.
Sa1760 Organ-Specific Migration of CD133+ Stem Cells Triggered by CpG Motifs From DSS-Induced Colitis Mouse Model Gabor 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 regenerative stem 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 of bacterial or viral DNA. Tissue damage is well characterized by increased levels of cell free DNA (cfDNA). Here we hypothesized, that CpG motifs of DNA from DSS-induced mouse may 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 colorectal intraepithelial CD133 protein positive cell number in healthy mice after the injection of DNA isolated from DSS-treated mouse plasma. Materials and methods: Mouse colitis model was established by treatment with 1% DSS, then cfDNA was isolated from DSS-treated animals and was injected into healthy mice. Peripherial blood samples were taken 1, 2, 4 and 24 hours after the treatment and the mRNA expression of CD133, TLR9 and inflammatory cascade members (such as IL-6, TNF-α) was measured by RT-PCR, where beta-2-microglobulin (B2M) was used as reference gene. From formalin fixed and paraffin embedded tissue samples (colon, spleen, kidney, lung and skin) of the treated animals 4 μm thick sections were cut and immunostained for CD133 polyclonal antibody (Abcam, Cambridge, UK). Then the slides were digitalized and analyzed by digital microscopy. Results: After 2 hours of the DNA injection the expression of all analyzed markers increased significantly in periferial blood 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 colonic epithelium showed increased number of goblet cells, as a sign of elevated regeneration. In contrast, no considerable morphological alteration or stem cell influx could be observed in
S-321
AGA Abstracts
AGA Abstracts
Oct4 Directs TGF-Beta Signaling in Embryonic Stem Cells Alan C. Mullen, Jamie J. Newman, David A. Orlando, Richard A. Young