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CD117 Identifies a Colonic Paneth-Like Cell That Contributes to the Stem Cell Niche and Regulates Crypt Homeostasis
907 Fasting Alters Cell Cycle Status in Both Rapidly and Slowly Cycling Intestinal Stem Cells Camilla A. Richmond, Bristol Brandt, Diana L. Carlone, Robert K. Montgomery, David T. Breault Mean (SEM) or median (IQR) shown for each treatment group. *Pre treatment was significantly different to post treatment using paired-t test or signed rank test (p-values 0.05-0.10 shown in parentheses, *p<0.05, **p<0.01, ***p<0.001). P-values for ANOVA across the four treatment groups for pre/post treatment were not significant.
Fasting is associated with dramatic physiologic changes designed to conserve energy and preserve systems in a dormant state, ensuring the resumption of normal homeostasis upon re-feeding. In the intestine, these changes give rise to decreased crypt cell proliferation and increased apoptosis resulting in villus atrophy. The effect of fasting on rapidly and slowly cycling intestinal stem cells (ISCs), required for intestinal maintenance and regeneration respectively, is not known. Given historical data, we hypothesized that both ISC populations would either enter or remain in a quiescent state and be resistant to apoptosis upon fasting. To investigate the role of rapidly cycling ISCs, we used Lgr5-GFP mice and used mTertGFP and mTert-CreER::R26R(LacZ) strains to study slowly cycling ISCs. Adult mice from each genotype were ad libitum fed (control) or fasted for 48h. Compared with controls, fasted animals lost 22% of their body weight, demonstrated a 15% decrease in villus height (p<0.0001) and a 23% decrease in villus cell number (p<0.0001), consistent with previous observations. Immunofluorescent analysis of control vs. fasted Lgr5 reporter mice revealed a dramatic decrease in Lgr5+Ki67+ cells (76% vs. 45%, p=0.02), indicating their exit from the cell cycle. Furthermore, analysis of Lgr5+ cells co-expressing activated caspase3 (a marker for apoptosis) in control vs. fasted mice revealed a trend towards an increase (0.5% vs. 2.6%, p=0.06) in the number of rapidly cycling ISCs undergoing apoptosis during fasting. In contrast to the above findings, analysis of slowly cycling ISCs using mTert reporter mice revealed a 10-fold induction in the number of mTert-GFP+ cells in fasted mice compared to controls (0.13% vs. 1.87%, p=0.03), which returned to near baseline levels after 24h of re-feeding (0.31%). These results were further supported by lineage-tracing analysis, which demonstrated a 7-fold increase in the percentage of mTert-CreER LacZ-marked crypts after fasting (control 0.012% vs. fasted 0.073%). We also observed a striking 10-fold induction in the number of actively cycling mTert-GFP+Ki67+ cells after fasting (5% vs. 53%, p= 0.01). In contrast to Lgr5-expressing cells, analysis of 150 mTert-GFP+ cells for co-expression with activated caspase3 revealed none to be undergoing programmed cell death. In conclusion, slowly cycling ISCs are induced to enter the cell cycle in response to fasting and are resistant to apoptosis. In contrast, rapidly cycling Lgr5+ ISCs largely exit the cell cycle and may undergo apoptosis at a higher rate. These data provide further insight into the role of slowly cycling ISCs and raise important questions about the mechanisms regulating intestinal homeostasis, suggesting that slowly cycling ISCs are poised to contribute to intestinal regeneration following physiologic stress.
904 Distribution of Plasma T and B Cells Differs in Children With Irritable Bowel Syndrome (IBS) vs. Healthy Children and Correlates With Abdominal Pain Robert J. Shulman, Michele Mariscalco, C. W. Smith, Margaret Heitkemper Background: Recent data from adult IBS patients suggest that the disorder is characterized, in part, by alterations in blood immune markers associated with low grade gastrointestinal mucosal inflammation. Whether children with IBS exhibit alterations in plasma lymphocytes and monocytes, and if so, whether they relate to abdominal pain symptoms is unknown. Methods: Premenarchal girls ages 7-12 yrs. were studied. Girls with IBS met Pediatric Rome III criteria. Healthy girls had no history of chronic abdominal pain complaints or chronic diseases. Both groups completed 14-day diaries which captured: number of abdominal pain episodes, pain severity (0-10 scale), mean maximum pain severity, whether a pain episode interfered with daily activities (yes/no), stool frequency, and stool form (Bristol Stool Form Scale). Freshly isolated blood was analyzed for surface T and B and monocyte cell markers using tagging with fluorescent markers and flow cytometry. Results: 13 IBS and 12 healthy girls were studied. Mean ages (SD) did not differ: 9.6 ± 1.5 vs 9.2 ± 1.6 yrs. (IBS vs Healthy, respectively). As expected, pain measures were significantly greater in the IBS group (P < 0.0004) except for pain interference with activity (P = 0.08). There were no differences between groups in stooling frequency or form. Percent of total lymphocytes (CD4+, CD8+, CD19+ combined) and monocytes (CD14+) did not differ between IBS and healthy girls. The mean percentage of CD8+ cells was higher in IBS vs healthy girls (30 ± 4 vs 25 ± 4, respectively; P = 0.012) whereas the percentage of CD19+ cells was lower (20 ± 5 vs 27 ± 4, respectively; P < 0.0004). There were no differences between groups in the percentages of CD4+ and CD14+ cells. There was a strong inverse correlation between the percentage of CD19+ cells and number of pain episodes (P < 0.0004, r2 = -0.37), pain severity (P = 0.009, r2 = 0.25), and mean maximum pain severity (P = 0.002, r2 = -0.36), but no relationship with pain interfering with activities. There was no relationship between cell markers and stooling characteristics. Conclusions: 1) Girls with IBS demonstrate alterations in the distribution of plasma T and B cells compared with healthy girls; 2) There is a strong inverse association between percentages of CD19+ cells and abdominal pain symptoms. These data suggest that an altered immune profile may play a role in abdominal pain symptoms in girls with IBS.
908 Prostaglandin E2 Promotes Mouse Colonic Organoid Growth and Human Colonic Crypt Stem/Progenitor Cell Proliferation Anastasia Sobolewski, Alexander Lambert, Ahmed El Hadi, Alyson Parris, Natalia Wharton, Kevin Sargen, Alison Prior, Richard Wharton, Christopher Speakman, Michael P. Lewis, Mark Williams
906
BACKGROUND: The emergence of intestinal inflammation as a major risk factor for colon cancer and the demonstration of a central role for intestinal stem cells in carcinogenesis are two of the most exciting advances in the last few years. Epidemiology, genetic studies, and an intestinal spheroid culture system have demonstrated a connection between the inflammatory mediator prostaglandin E2 (PGE2), Wnt signalling and the maintenance of intestinal stem cells. As such, pharmacological manipulation of this pathway still holds great promise for chemoprevention and anti-inflammatory strategies. The aim of this study was to investigate the expression of PGE2 receptor (EP) subtypes and assess the contribution of PGE2 to the maintenance and regeneration of the intestinal epithelium. METHODS: Mouse colon or tissue biopsies obtained at sigmoidoscopy (Ethical approval) were immediately fixed or processed for crypt isolation and placed into 3D culture in the presence of Wnt/ R-spondin-1 and other growth factors. Cultured human colonic crypts and mouse colonic organoids were subjected to immunofluorescence for detection of LGR5, OLFM4, EP1-4, Ki67 and BrdU. Fluorescence labelling was visualised in 3D by confocal microscopy. RESULTS: EP 1-4 receptors are expressed on basal membranes of the human (n=3) and mouse crypt epithelium (n=4). EP 1,2 and 3 demonstrated a gradient of receptor expression with highest expression at the crypt-base and lowest at the surface epithelium of microdissected and
CKit/CD117 Identifies a Colonic Paneth-Like Cell That Contributes to the Stem Cell Niche and Regulates Crypt Homeostasis Michael E. Rothenberg, Ysbrand Nusse, Tomer Kalisky, John Lee, Piero D. Dalerba, Agnieszka Czechowicz, Ferenc Scheeren, Subhash Kulkarni, Neethan A. Lobo, Sopheak Sim, Philip Beachy, Pankaj J. Pasricha, Stephen Quake, Michael F. Clarke Background and Aims: Lgr5+ stem cells in the small intestinal and colonic crypts help drive the continuous self-renewal of the epithelium. In the small intestine, Paneth cells are adjacent to the Lgr5+ stem cells and function as niche cells, providing important secreted and transmembrane regulatory cues such as Wnt agonists, Notch agonists, and EGF receptor agonists. Interestingly, the colon does not have Paneth cells, yet in-vitro growth of colonic Lgr5+ stem cells, and In-Vivo colonic epithelial homeostasis, depends on many of the same molecular signals as in the small intestine. The source of these signals has remained obscure. We hypothesize the following: 1) a colonic Paneth-like niche cell exists, 2) it supports the Lgr5+ stem cells, 3) it has a distinct transcriptional signature, and 4) it has a surface
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AGA Abstracts
AGA Abstracts
phenotype that distinguishes it from surrounding colonocytes. Methods: We conducted multicolor fluorescence-activated cell sorting (FACS) with widely-available surface markers to isolate different colon crypt subregions from dissociated murine colonic epithelium. We validated our FACS isolation using several distinct modalities of gene expression analysis. We then utilized the novel technique of highly multiplexed single-cell gene expression analysis with qRT-PCR, followed by hierarchical clustering analysis, to identify and characterize distinct cell types. After this approach identified a colonic Paneth-like cell, we screened for surface markers that could isolate this cell type. We subsequently characterized this cell type using immunostaining, FACS, in-vitro organoid culture, and In-Vivo studies on murine small intestine and colon. Results: Colonic goblet cells exhibit striking heterogeneity at the single-cell level and contain a distinct Dll1+Dll4+ subpopulation that is CD24-high and expresses EGF—-similar to Paneth cells. cKit/CD117 specifically marks these cells (in the colon) and Paneth cells (in the small intestine). Like Paneth cells, cKit+ colonocytes are interdigitated between Lgr5+ stem cells. When isolated from mouse colon, they promote organoid formation of Lgr5+ cells, which express Kitl/SCF, the ligand for cKit. When organoids are depleted of cKit+ cells with a toxin-conjugated antibody, organoid formation decreases significantly. In Vivo, injection of a cKit blocking antibody leads to depletion of Paneth cells and colonic goblet cells. Furthermore, the cKit+ population is regulated by Notch signaling, since treatment of mice with a gamma-secretase inhibitor increases the number of cKit+ cells. Conclusions: cKit marks small intestinal Paneth cells and also identifies a novel colonic Paneth-like niche cell. This cell is regulated by Notch signaling and supports Lgr5+ cells. Since adjacent Lgr5+ cells express Kitl/SCF, bidirectional signaling between Lgr5+ cells and niche cells may occur.
Fasting is associated with dramatic physiologic changes designed to conserve energy and preserve systems in a dormant state, ensuring the resumption of normal homeostasis upon re-feeding. In the intestine, these changes give rise to decreased crypt cell proliferation and increased apoptosis resulting in villus atrophy. The effect of fasting on rapidly and slowly cycling intestinal stem cells (ISCs), required for intestinal maintenance and regeneration respectively, is not known. Given historical data, we hypothesized that both ISC populations would either enter or remain in a quiescent state and be resistant to apoptosis upon fasting. To investigate the role of rapidly cycling ISCs, we used Lgr5-GFP mice and used mTertGFP and mTert-CreER::R26R(LacZ) strains to study slowly cycling ISCs. Adult mice from each genotype were ad libitum fed (control) or fasted for 48h. Compared with controls, fasted animals lost 22% of their body weight, demonstrated a 15% decrease in villus height (p<0.0001) and a 23% decrease in villus cell number (p<0.0001), consistent with previous observations. Immunofluorescent analysis of control vs. fasted Lgr5 reporter mice revealed a dramatic decrease in Lgr5+Ki67+ cells (76% vs. 45%, p=0.02), indicating their exit from the cell cycle. Furthermore, analysis of Lgr5+ cells co-expressing activated caspase3 (a marker for apoptosis) in control vs. fasted mice revealed a trend towards an increase (0.5% vs. 2.6%, p=0.06) in the number of rapidly cycling ISCs undergoing apoptosis during fasting. In contrast to the above findings, analysis of slowly cycling ISCs using mTert reporter mice revealed a 10-fold induction in the number of mTert-GFP+ cells in fasted mice compared to controls (0.13% vs. 1.87%, p=0.03), which returned to near baseline levels after 24h of re-feeding (0.31%). These results were further supported by lineage-tracing analysis, which demonstrated a 7-fold increase in the percentage of mTert-CreER LacZ-marked crypts after fasting (control 0.012% vs. fasted 0.073%). We also observed a striking 10-fold induction in the number of actively cycling mTert-GFP+Ki67+ cells after fasting (5% vs. 53%, p= 0.01). In contrast to Lgr5-expressing cells, analysis of 150 mTert-GFP+ cells for co-expression with activated caspase3 revealed none to be undergoing programmed cell death. In conclusion, slowly cycling ISCs are induced to enter the cell cycle in response to fasting and are resistant to apoptosis. In contrast, rapidly cycling Lgr5+ ISCs largely exit the cell cycle and may undergo apoptosis at a higher rate. These data provide further insight into the role of slowly cycling ISCs and raise important questions about the mechanisms regulating intestinal homeostasis, suggesting that slowly cycling ISCs are poised to contribute to intestinal regeneration following physiologic stress.
904 Distribution of Plasma T and B Cells Differs in Children With Irritable Bowel Syndrome (IBS) vs. Healthy Children and Correlates With Abdominal Pain Robert J. Shulman, Michele Mariscalco, C. W. Smith, Margaret Heitkemper Background: Recent data from adult IBS patients suggest that the disorder is characterized, in part, by alterations in blood immune markers associated with low grade gastrointestinal mucosal inflammation. Whether children with IBS exhibit alterations in plasma lymphocytes and monocytes, and if so, whether they relate to abdominal pain symptoms is unknown. Methods: Premenarchal girls ages 7-12 yrs. were studied. Girls with IBS met Pediatric Rome III criteria. Healthy girls had no history of chronic abdominal pain complaints or chronic diseases. Both groups completed 14-day diaries which captured: number of abdominal pain episodes, pain severity (0-10 scale), mean maximum pain severity, whether a pain episode interfered with daily activities (yes/no), stool frequency, and stool form (Bristol Stool Form Scale). Freshly isolated blood was analyzed for surface T and B and monocyte cell markers using tagging with fluorescent markers and flow cytometry. Results: 13 IBS and 12 healthy girls were studied. Mean ages (SD) did not differ: 9.6 ± 1.5 vs 9.2 ± 1.6 yrs. (IBS vs Healthy, respectively). As expected, pain measures were significantly greater in the IBS group (P < 0.0004) except for pain interference with activity (P = 0.08). There were no differences between groups in stooling frequency or form. Percent of total lymphocytes (CD4+, CD8+, CD19+ combined) and monocytes (CD14+) did not differ between IBS and healthy girls. The mean percentage of CD8+ cells was higher in IBS vs healthy girls (30 ± 4 vs 25 ± 4, respectively; P = 0.012) whereas the percentage of CD19+ cells was lower (20 ± 5 vs 27 ± 4, respectively; P < 0.0004). There were no differences between groups in the percentages of CD4+ and CD14+ cells. There was a strong inverse correlation between the percentage of CD19+ cells and number of pain episodes (P < 0.0004, r2 = -0.37), pain severity (P = 0.009, r2 = 0.25), and mean maximum pain severity (P = 0.002, r2 = -0.36), but no relationship with pain interfering with activities. There was no relationship between cell markers and stooling characteristics. Conclusions: 1) Girls with IBS demonstrate alterations in the distribution of plasma T and B cells compared with healthy girls; 2) There is a strong inverse association between percentages of CD19+ cells and abdominal pain symptoms. These data suggest that an altered immune profile may play a role in abdominal pain symptoms in girls with IBS.
908 Prostaglandin E2 Promotes Mouse Colonic Organoid Growth and Human Colonic Crypt Stem/Progenitor Cell Proliferation Anastasia Sobolewski, Alexander Lambert, Ahmed El Hadi, Alyson Parris, Natalia Wharton, Kevin Sargen, Alison Prior, Richard Wharton, Christopher Speakman, Michael P. Lewis, Mark Williams
906
BACKGROUND: The emergence of intestinal inflammation as a major risk factor for colon cancer and the demonstration of a central role for intestinal stem cells in carcinogenesis are two of the most exciting advances in the last few years. Epidemiology, genetic studies, and an intestinal spheroid culture system have demonstrated a connection between the inflammatory mediator prostaglandin E2 (PGE2), Wnt signalling and the maintenance of intestinal stem cells. As such, pharmacological manipulation of this pathway still holds great promise for chemoprevention and anti-inflammatory strategies. The aim of this study was to investigate the expression of PGE2 receptor (EP) subtypes and assess the contribution of PGE2 to the maintenance and regeneration of the intestinal epithelium. METHODS: Mouse colon or tissue biopsies obtained at sigmoidoscopy (Ethical approval) were immediately fixed or processed for crypt isolation and placed into 3D culture in the presence of Wnt/ R-spondin-1 and other growth factors. Cultured human colonic crypts and mouse colonic organoids were subjected to immunofluorescence for detection of LGR5, OLFM4, EP1-4, Ki67 and BrdU. Fluorescence labelling was visualised in 3D by confocal microscopy. RESULTS: EP 1-4 receptors are expressed on basal membranes of the human (n=3) and mouse crypt epithelium (n=4). EP 1,2 and 3 demonstrated a gradient of receptor expression with highest expression at the crypt-base and lowest at the surface epithelium of microdissected and
CKit/CD117 Identifies a Colonic Paneth-Like Cell That Contributes to the Stem Cell Niche and Regulates Crypt Homeostasis Michael E. Rothenberg, Ysbrand Nusse, Tomer Kalisky, John Lee, Piero D. Dalerba, Agnieszka Czechowicz, Ferenc Scheeren, Subhash Kulkarni, Neethan A. Lobo, Sopheak Sim, Philip Beachy, Pankaj J. Pasricha, Stephen Quake, Michael F. Clarke Background and Aims: Lgr5+ stem cells in the small intestinal and colonic crypts help drive the continuous self-renewal of the epithelium. In the small intestine, Paneth cells are adjacent to the Lgr5+ stem cells and function as niche cells, providing important secreted and transmembrane regulatory cues such as Wnt agonists, Notch agonists, and EGF receptor agonists. Interestingly, the colon does not have Paneth cells, yet in-vitro growth of colonic Lgr5+ stem cells, and In-Vivo colonic epithelial homeostasis, depends on many of the same molecular signals as in the small intestine. The source of these signals has remained obscure. We hypothesize the following: 1) a colonic Paneth-like niche cell exists, 2) it supports the Lgr5+ stem cells, 3) it has a distinct transcriptional signature, and 4) it has a surface
S-159
AGA Abstracts
AGA Abstracts
phenotype that distinguishes it from surrounding colonocytes. Methods: We conducted multicolor fluorescence-activated cell sorting (FACS) with widely-available surface markers to isolate different colon crypt subregions from dissociated murine colonic epithelium. We validated our FACS isolation using several distinct modalities of gene expression analysis. We then utilized the novel technique of highly multiplexed single-cell gene expression analysis with qRT-PCR, followed by hierarchical clustering analysis, to identify and characterize distinct cell types. After this approach identified a colonic Paneth-like cell, we screened for surface markers that could isolate this cell type. We subsequently characterized this cell type using immunostaining, FACS, in-vitro organoid culture, and In-Vivo studies on murine small intestine and colon. Results: Colonic goblet cells exhibit striking heterogeneity at the single-cell level and contain a distinct Dll1+Dll4+ subpopulation that is CD24-high and expresses EGF—-similar to Paneth cells. cKit/CD117 specifically marks these cells (in the colon) and Paneth cells (in the small intestine). Like Paneth cells, cKit+ colonocytes are interdigitated between Lgr5+ stem cells. When isolated from mouse colon, they promote organoid formation of Lgr5+ cells, which express Kitl/SCF, the ligand for cKit. When organoids are depleted of cKit+ cells with a toxin-conjugated antibody, organoid formation decreases significantly. In Vivo, injection of a cKit blocking antibody leads to depletion of Paneth cells and colonic goblet cells. Furthermore, the cKit+ population is regulated by Notch signaling, since treatment of mice with a gamma-secretase inhibitor increases the number of cKit+ cells. Conclusions: cKit marks small intestinal Paneth cells and also identifies a novel colonic Paneth-like niche cell. This cell is regulated by Notch signaling and supports Lgr5+ cells. Since adjacent Lgr5+ cells express Kitl/SCF, bidirectional signaling between Lgr5+ cells and niche cells may occur.