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Su1736 Live Imaging of Single Cell Reveals Single Stem Cell Dynamics in an Organoid Derived From Murine Small Intestine
AGA Abstracts
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.
intestine and cancer cells were evaluated. As stem cells are under-represented in duodenal biopsies when compared to differentiated cells, we used stem-progenitor enriched cells from intestinal crypts to prepare RNA. Crypts from biopsies of active celiac patients (N=3) and healthy controls (N=2) were isolated by laser capture microscopy (LCM). The genes expression was evaluated by quantitative RT-PCR array, according to the manufacturer protocol (Stem cell signaling Profiler Array SABiosciences-Qiagen). Results: Our results indicated that only a few pathways, involved in stem cell differentiation and proliferation, were altered. Namely: FGF, Tgf-beta/BMP and Notch were upregulated. Furthermore, genes involved in regulating the Epithelial Mesenchymal Transition (EMT) like ZEB2 and LIFR were downregulated compared to healthy control. Interestingly, the Wnt pathway was unchanged as regards of transcription. Conclusions: Our data suggest that in celiac patients the immature compartment is expanded. Upregulation of Notch and FGF might be responsible for expansion of this compartment and involved in crypt hyperplasia. Further studies are required to evaluate this hypothesis. On the contrary the stem cell compartment is not expanded based on absence of Wnt pathway upregulation. Although celiac duodenal biopsies have elevated TGF-beta expression compared to normal control, this cytokine appears to be inversely regulated in stem/progenitor cells compartment from celiac disease patients. Interestingly Tgf-beta promotes EMT and differentiation. Further ZEB2 and LIFR, key components of EMT, were also downregulated suggesting that EMT might be critical during the acute phase of the disease. Confirmation of these preliminary results is necessary to provide more insights on the contribution of ISC network pathways to the onset and maintenance of the celiac disease histopathology.
Su1736 Live Imaging of Single Cell Reveals Single Stem Cell Dynamics in an Organoid Derived From Murine Small Intestine Nobukatsu Horita, Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Yoshihito Kano, Xiu Zheng, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe Background & Aim: Many advances have been reported in the mouse adult intestinal stem cell in recent years. Two kinds of stem cell population are advocated: +4 cell, located directly above the Paneth cell, slow cycling and Crypt base columnar (CBC) cell, expressing Lgr5, rapidly cycling. A recent study has described 3-dimentional small intestinal organoids can be generated from a single Lgr5+ stem cell. However, the stem cell dynamics in a crypt remains not to be fully elucidated, because single stem cell has not been visualized in live. We hypothesized that stem cell dynamics and relationship between stem cell populations may able to be explored by live imaging in vitro culture, using fluorescing cells. Methods: Mouse small intestine were immediately processed for crypt isolation and placed into 3D matrigel-based culture in according with previous report. Lentivirus encoding mCherry gene was mixed with matrigel to directly infect the organoids. The localization of mCherry positive cells in whole organoids were analyzed by confocal microscope and time-lapse imaging microscope. The cell types of mCherry positive cells were identified by the immunostaining with OLFM4 and Lysozyme. Results: We have established a lentiviral transduction system to detect mCherry protein expression for a long time, 50 weeks after transduction. All kinds of cells in organoid were able to express mCherry protein and be detected by confocal microscope, which can identify the every single cell at high resolution in a whole organoid. Moreover, we found the crypt in which only single mCherry positive was continuously located. Single mCherry positive cells was co-localized with OLFM4, suggesting that single mCherry positive cell might be a stem cell in a crypt. Furthermore, we found that single mCherry positive cell was differentiated to a Paneth cell by the immunostaining with Lysozyme. Time-lapse imaging showed that a single mCherry positive stem cell in a crypt was divided into the other CBC cells and supplied the cell into the villi. Conclusions: Live imaging of single cell in a crypt revealed the stem cell division and maintenance in live. Labeling single stem cell by lentivirus infection might be useful to elucidate the behavior of intestinal stem cells and the relationship between two stem cell populations.
Su1739 Mice With Allelic Variation on Chromosome 11 Respond Differentially to Citrobacter Rodentium Infection Ishfaq Ahmed, Rao Papineni, Kenneth Bradley, Shrikant Anant, Steven LeVine, Shahid Umar Background: B6.CAST.11M mice contain a CAST/Ei region on chromosome 11 on an otherwise C57BL/6 genetic background. Genetic variation within an 8.3Mb region on mouse chromosome 11 controls host response to anthrax lethal toxin and resistance to infection by the Sterne strain of Bacillus anthracis. Citrobacter rodentium (CR) induces transmissible murine colonic hyperplasia and varying degrees of inflammation depending on the genetic background. Aim: To investigate whether allelic variation on chromosome 11 may contribute towards a differential response to CR infection. Methods: Both C57BL/6 (C57) and B6.CAST.11M (11M) mice were infected with CR. Colonic crypts and crypt-denuded lamina propria (CLP) were isolated and various molecular, biochemical or immunohistochemical techniques were employed for analysis. Results: Following CR infection, C57 mice exhibited significant crypt hyperplasia as revealed by Ki-67 staining at day 9 which peaked by day 12 and plateaued by day 19 that coincided with nuclear accumulation of β-catenin and its downstream target cyclinD1 at these time points. In addition, NF- κB activity and p65276/ p65536 subunit expression increased significantly at days 9 and 12 with declining trend at day 19 that correlated with downstream target CXCL-1 expression. C57 mice also exhibited significant disruption in intestinal barrier as revealed by increased levels of serum FITCDextran that accompanied recruitment of CD3+ T-cells and F4/80+ macrophages to the sub-epithelial regions. In 11M mice, Ki-67 staining and crypt lengths at days 12 and 19 along with nuclear β-catenin and cyclinD1 were two-fold higher than those recorded in C57 mice. 11M mice however, did not exhibit any increase in NF- κB activity, CXCL-1 expression or serum FITC-Dextran levels at these time points despite adequate bacterial binding to the colonic mucosa. During measurement of Reactive Oxygen Species (ROS) in the gut, robust elevation in ROS activity was recorded at days 9 and 12 post-infection in C57 mice with declining trend at day 19. In 11M mice, ROS activity was low at day 9 but increased significantly at day 12 and was almost two-fold higher at day 19. Relative expression of heat shock protein 75 (HSP75), an antagonist of ROS, increased significantly in the crypts of either mice at days 9-12 compared to uninfected control. In CLP however, dramatic decrease in HSP75 levels at days 9 and 12 in C57 mice correlated with increased ROS activity. In 11M mice, HSP75 levels at day 9 were elevated compared to uninfected controls while a significant decrease at days 12 and 19 correlated with increased ROS activity. Conclusions: 1. B6.CAST.11M mice respond differentially to CR infection compared to C57BL/6 mice. 2. ROS generation is predominantly stromal and may be regulated by the contribution of molecular determinants at chromosome 11.
Su1737 Modulation of Stemness in a Human Intestinal Epithelial Crypt Cell Line Amel Guezguez, Yannick D. Benoit, Blanche C. Senicourt, Nuria Basora, Jean-Francois Beaulieu Background: The intestinal epithelium contains a rapidly proliferating and perpetually differentiating epithelium. Stem cells are located in the lower third of the crypts and give rise to transit amplifying cells that can differentiate into the 4 major epithelial cell types. The study of adult gastrointestinal tract stem cells has progressed rapidly with the recent discovery of several putative stem cell markers such as BMI1 and LGR5 and the characterization of the niche-related Wnt, BMP and Notch signaling pathways. HIEC-6 cells, originally derived from the human fetal intestine, were recently shown to express a number of cell markers previously reported in the intestinal stem cell compartment such as BMI1, DCAMKL-1 and Musashi. Aim: In the present study, we investigated the expression of a panel of intestinal stem cell markers in HIEC-6 cells under normal culture parameters as well as under conditions that mimic the normal stem cell microenvironment. Methods: HIEC were grown under standard conditions with 4% FBS containing medium +/- 5ng/ml Wnt3a, 10 ng/ml R-spondin and/or 100 ng/ml noggin for 48h. Intestinal stem cell markers were assessed by qPCR and Western blot. Wnt pathway activation and cell proliferation were evaluated by TOPFlash assays and BrdU incorporation, respectively. Results: Control HIEC cells were found to express significant amounts of BMI1 and HOPX and low amounts of LGR5 and PHLDA1 and, consistent with these observation, relatively low β-catenin/TCF activity and high BMP activity were observed. Stimulation of HIEC cells with the Wnt agonists induced β-catenin/ TCF activity and expression of the Wnt target genes Cyclin D2 and LGR5. Dual activation of the Wnt pathway and inhibition of the BMP pathway resulted in further increases of LGR5 and PHLDA1 and a decrease of BMI1 and HOPX cellular levels. Cell proliferation was not affected. Conclusion: These findings demonstrate that established cultures of normal human intestinal epithelial crypt cells can be prompted toward a stem-like cell phenotype by modulating a few key cellular pathways. This model should be useful to further define the human intestinal stem cell niche.
Su1740 The Intestinal Stem Cells Marker LGR5 Is Regulated by Nutrients Availability and Differentiation in a Colon Cancer Model: Implications for Metabolism Based Therapies Valentina Tesori, Maria Ausiliatrice Puglisi, Giovanni Gasbarrini, Antonio Gasbarrini BACKGROUND: The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) is one of the best characterized adult stem cell marker in the gut. Recent evidences point toward a role for intestinal stem cells (ISCS) as nutrient sensors, regulating organ growth in response to food availability. Caloric restriction is protective against tumors, and this is of considerable importance in the case of colon cancer. AIM: Aim of the present study was to investigate the role of LGR5 in Colon Cancer differentiation, correlating it with nutrients availability and glucose metabolism. MATHERIALS AND METHODS: Gene expression was assessed by qRT-PCR. Differentiation was performed administrating sodium butyrate and measuring alkaline phosphatase (ALP) activity. Protein expression was assessed by western blotting. RESULTS: During differentiation, we found an increase in the LGR5 mRNA, peaking at 48 hours, in Caco2 and HT-29 CC cells; also the expression of b-catenin, a key regulator of LGR5, was upregulated; interestingly, the mitochondrial enzyme PDHA1 is upregulated during differentiation, showing that differentiated cells are less glycolytic than undifferentiated ones and rely more on oxidative phosphorylation. Since it has recently been discovered that differentiation is associated with a change in glucose metabolism, we investigated whether glucose could directly regulate LGR5; indeed, in low glucose conditions (1g/L) LGR5 was downregulated at the mRNA and protein level; moreover, in low glucose, CC cells shown an higher level of differentiation, versus high glucose medium, especially at 24
Su1738 Expression Analysis of Intestinal Stem Cell Signaling Pathways in Acute Celiac Disease Stefania Senger, Anna Sapone, Giuseppe Mazzarella, Alessio Fasano Background: Celiac disease is an autoimmune disease triggered by ingestion of gluten, the key proteic component of wheat, in genetically predisposed individuals. The intestinal mucosa presents lesions with varying degrees of inflammation secondary to the autoimmune insult characterized by villous atrophy and crypts hyperplasia. Aims: The current study is aimed at understanding the biochemical response to inflammation of the intestinal stem cell compartment (ISC) during the acute phase of celiac disease. Methods: The expression profile of 86 genes previously identified to be involved in stem cell signaling pathways in mouse
AGA Abstracts
S-464
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.
intestine and cancer cells were evaluated. As stem cells are under-represented in duodenal biopsies when compared to differentiated cells, we used stem-progenitor enriched cells from intestinal crypts to prepare RNA. Crypts from biopsies of active celiac patients (N=3) and healthy controls (N=2) were isolated by laser capture microscopy (LCM). The genes expression was evaluated by quantitative RT-PCR array, according to the manufacturer protocol (Stem cell signaling Profiler Array SABiosciences-Qiagen). Results: Our results indicated that only a few pathways, involved in stem cell differentiation and proliferation, were altered. Namely: FGF, Tgf-beta/BMP and Notch were upregulated. Furthermore, genes involved in regulating the Epithelial Mesenchymal Transition (EMT) like ZEB2 and LIFR were downregulated compared to healthy control. Interestingly, the Wnt pathway was unchanged as regards of transcription. Conclusions: Our data suggest that in celiac patients the immature compartment is expanded. Upregulation of Notch and FGF might be responsible for expansion of this compartment and involved in crypt hyperplasia. Further studies are required to evaluate this hypothesis. On the contrary the stem cell compartment is not expanded based on absence of Wnt pathway upregulation. Although celiac duodenal biopsies have elevated TGF-beta expression compared to normal control, this cytokine appears to be inversely regulated in stem/progenitor cells compartment from celiac disease patients. Interestingly Tgf-beta promotes EMT and differentiation. Further ZEB2 and LIFR, key components of EMT, were also downregulated suggesting that EMT might be critical during the acute phase of the disease. Confirmation of these preliminary results is necessary to provide more insights on the contribution of ISC network pathways to the onset and maintenance of the celiac disease histopathology.
Su1736 Live Imaging of Single Cell Reveals Single Stem Cell Dynamics in an Organoid Derived From Murine Small Intestine Nobukatsu Horita, Kiichiro Tsuchiya, Shuji Hibiya, Keita Fukushima, Yoshihito Kano, Xiu Zheng, Ryuichi Okamoto, Tetsuya Nakamura, Mamoru Watanabe Background & Aim: Many advances have been reported in the mouse adult intestinal stem cell in recent years. Two kinds of stem cell population are advocated: +4 cell, located directly above the Paneth cell, slow cycling and Crypt base columnar (CBC) cell, expressing Lgr5, rapidly cycling. A recent study has described 3-dimentional small intestinal organoids can be generated from a single Lgr5+ stem cell. However, the stem cell dynamics in a crypt remains not to be fully elucidated, because single stem cell has not been visualized in live. We hypothesized that stem cell dynamics and relationship between stem cell populations may able to be explored by live imaging in vitro culture, using fluorescing cells. Methods: Mouse small intestine were immediately processed for crypt isolation and placed into 3D matrigel-based culture in according with previous report. Lentivirus encoding mCherry gene was mixed with matrigel to directly infect the organoids. The localization of mCherry positive cells in whole organoids were analyzed by confocal microscope and time-lapse imaging microscope. The cell types of mCherry positive cells were identified by the immunostaining with OLFM4 and Lysozyme. Results: We have established a lentiviral transduction system to detect mCherry protein expression for a long time, 50 weeks after transduction. All kinds of cells in organoid were able to express mCherry protein and be detected by confocal microscope, which can identify the every single cell at high resolution in a whole organoid. Moreover, we found the crypt in which only single mCherry positive was continuously located. Single mCherry positive cells was co-localized with OLFM4, suggesting that single mCherry positive cell might be a stem cell in a crypt. Furthermore, we found that single mCherry positive cell was differentiated to a Paneth cell by the immunostaining with Lysozyme. Time-lapse imaging showed that a single mCherry positive stem cell in a crypt was divided into the other CBC cells and supplied the cell into the villi. Conclusions: Live imaging of single cell in a crypt revealed the stem cell division and maintenance in live. Labeling single stem cell by lentivirus infection might be useful to elucidate the behavior of intestinal stem cells and the relationship between two stem cell populations.
Su1739 Mice With Allelic Variation on Chromosome 11 Respond Differentially to Citrobacter Rodentium Infection Ishfaq Ahmed, Rao Papineni, Kenneth Bradley, Shrikant Anant, Steven LeVine, Shahid Umar Background: B6.CAST.11M mice contain a CAST/Ei region on chromosome 11 on an otherwise C57BL/6 genetic background. Genetic variation within an 8.3Mb region on mouse chromosome 11 controls host response to anthrax lethal toxin and resistance to infection by the Sterne strain of Bacillus anthracis. Citrobacter rodentium (CR) induces transmissible murine colonic hyperplasia and varying degrees of inflammation depending on the genetic background. Aim: To investigate whether allelic variation on chromosome 11 may contribute towards a differential response to CR infection. Methods: Both C57BL/6 (C57) and B6.CAST.11M (11M) mice were infected with CR. Colonic crypts and crypt-denuded lamina propria (CLP) were isolated and various molecular, biochemical or immunohistochemical techniques were employed for analysis. Results: Following CR infection, C57 mice exhibited significant crypt hyperplasia as revealed by Ki-67 staining at day 9 which peaked by day 12 and plateaued by day 19 that coincided with nuclear accumulation of β-catenin and its downstream target cyclinD1 at these time points. In addition, NF- κB activity and p65276/ p65536 subunit expression increased significantly at days 9 and 12 with declining trend at day 19 that correlated with downstream target CXCL-1 expression. C57 mice also exhibited significant disruption in intestinal barrier as revealed by increased levels of serum FITCDextran that accompanied recruitment of CD3+ T-cells and F4/80+ macrophages to the sub-epithelial regions. In 11M mice, Ki-67 staining and crypt lengths at days 12 and 19 along with nuclear β-catenin and cyclinD1 were two-fold higher than those recorded in C57 mice. 11M mice however, did not exhibit any increase in NF- κB activity, CXCL-1 expression or serum FITC-Dextran levels at these time points despite adequate bacterial binding to the colonic mucosa. During measurement of Reactive Oxygen Species (ROS) in the gut, robust elevation in ROS activity was recorded at days 9 and 12 post-infection in C57 mice with declining trend at day 19. In 11M mice, ROS activity was low at day 9 but increased significantly at day 12 and was almost two-fold higher at day 19. Relative expression of heat shock protein 75 (HSP75), an antagonist of ROS, increased significantly in the crypts of either mice at days 9-12 compared to uninfected control. In CLP however, dramatic decrease in HSP75 levels at days 9 and 12 in C57 mice correlated with increased ROS activity. In 11M mice, HSP75 levels at day 9 were elevated compared to uninfected controls while a significant decrease at days 12 and 19 correlated with increased ROS activity. Conclusions: 1. B6.CAST.11M mice respond differentially to CR infection compared to C57BL/6 mice. 2. ROS generation is predominantly stromal and may be regulated by the contribution of molecular determinants at chromosome 11.
Su1737 Modulation of Stemness in a Human Intestinal Epithelial Crypt Cell Line Amel Guezguez, Yannick D. Benoit, Blanche C. Senicourt, Nuria Basora, Jean-Francois Beaulieu Background: The intestinal epithelium contains a rapidly proliferating and perpetually differentiating epithelium. Stem cells are located in the lower third of the crypts and give rise to transit amplifying cells that can differentiate into the 4 major epithelial cell types. The study of adult gastrointestinal tract stem cells has progressed rapidly with the recent discovery of several putative stem cell markers such as BMI1 and LGR5 and the characterization of the niche-related Wnt, BMP and Notch signaling pathways. HIEC-6 cells, originally derived from the human fetal intestine, were recently shown to express a number of cell markers previously reported in the intestinal stem cell compartment such as BMI1, DCAMKL-1 and Musashi. Aim: In the present study, we investigated the expression of a panel of intestinal stem cell markers in HIEC-6 cells under normal culture parameters as well as under conditions that mimic the normal stem cell microenvironment. Methods: HIEC were grown under standard conditions with 4% FBS containing medium +/- 5ng/ml Wnt3a, 10 ng/ml R-spondin and/or 100 ng/ml noggin for 48h. Intestinal stem cell markers were assessed by qPCR and Western blot. Wnt pathway activation and cell proliferation were evaluated by TOPFlash assays and BrdU incorporation, respectively. Results: Control HIEC cells were found to express significant amounts of BMI1 and HOPX and low amounts of LGR5 and PHLDA1 and, consistent with these observation, relatively low β-catenin/TCF activity and high BMP activity were observed. Stimulation of HIEC cells with the Wnt agonists induced β-catenin/ TCF activity and expression of the Wnt target genes Cyclin D2 and LGR5. Dual activation of the Wnt pathway and inhibition of the BMP pathway resulted in further increases of LGR5 and PHLDA1 and a decrease of BMI1 and HOPX cellular levels. Cell proliferation was not affected. Conclusion: These findings demonstrate that established cultures of normal human intestinal epithelial crypt cells can be prompted toward a stem-like cell phenotype by modulating a few key cellular pathways. This model should be useful to further define the human intestinal stem cell niche.
Su1740 The Intestinal Stem Cells Marker LGR5 Is Regulated by Nutrients Availability and Differentiation in a Colon Cancer Model: Implications for Metabolism Based Therapies Valentina Tesori, Maria Ausiliatrice Puglisi, Giovanni Gasbarrini, Antonio Gasbarrini BACKGROUND: The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5) is one of the best characterized adult stem cell marker in the gut. Recent evidences point toward a role for intestinal stem cells (ISCS) as nutrient sensors, regulating organ growth in response to food availability. Caloric restriction is protective against tumors, and this is of considerable importance in the case of colon cancer. AIM: Aim of the present study was to investigate the role of LGR5 in Colon Cancer differentiation, correlating it with nutrients availability and glucose metabolism. MATHERIALS AND METHODS: Gene expression was assessed by qRT-PCR. Differentiation was performed administrating sodium butyrate and measuring alkaline phosphatase (ALP) activity. Protein expression was assessed by western blotting. RESULTS: During differentiation, we found an increase in the LGR5 mRNA, peaking at 48 hours, in Caco2 and HT-29 CC cells; also the expression of b-catenin, a key regulator of LGR5, was upregulated; interestingly, the mitochondrial enzyme PDHA1 is upregulated during differentiation, showing that differentiated cells are less glycolytic than undifferentiated ones and rely more on oxidative phosphorylation. Since it has recently been discovered that differentiation is associated with a change in glucose metabolism, we investigated whether glucose could directly regulate LGR5; indeed, in low glucose conditions (1g/L) LGR5 was downregulated at the mRNA and protein level; moreover, in low glucose, CC cells shown an higher level of differentiation, versus high glucose medium, especially at 24
Su1738 Expression Analysis of Intestinal Stem Cell Signaling Pathways in Acute Celiac Disease Stefania Senger, Anna Sapone, Giuseppe Mazzarella, Alessio Fasano Background: Celiac disease is an autoimmune disease triggered by ingestion of gluten, the key proteic component of wheat, in genetically predisposed individuals. The intestinal mucosa presents lesions with varying degrees of inflammation secondary to the autoimmune insult characterized by villous atrophy and crypts hyperplasia. Aims: The current study is aimed at understanding the biochemical response to inflammation of the intestinal stem cell compartment (ISC) during the acute phase of celiac disease. Methods: The expression profile of 86 genes previously identified to be involved in stem cell signaling pathways in mouse
AGA Abstracts
S-464