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264 Extracellular Matrix Directs Differentiation of Motor Neuronal Subtypes in Tissue Engineered Innervated Intestinal Smooth Muscle
weak expression of c-Kit emerges in cells with strong Lrig1 expression at the inner border of the circular muscle layer. For lineage tracing, tamoxifen was administrated at P0 when Lrig1 is only expressed in smooth muscle cells beneath the submucosa in the proximal colon. After 9 days, YFP immunoreactivity was detected in ICC-SMP. Conclusions: Lrig1 is found to regulate the development of ICC-DMP and ICC-SMP. In addition, loss of Lrig1 slowed gut motility, possibly due to perturbations in the ICC-DMP and ICC-SMP populations. By lineage tracing, smooth muscular cells give rise to Lrig1-expressing ICC-SMP.
Identification and Characterization of a Subset of Enteric "Glial" Cells As Neural Precursors Subhash Kulkarni, Cuiping Li, Maria-Adelaide Micci, Pankaj J. Pasricha Introduction: Recently, it has been shown that cells expressing the glial marker GFAP can make enteric neurons, raising the possibility that enteric glia can function as enteric neural precursor cells (ENPC). However, it is not known whether all or only a sub-population of enteric glia are ENPC. Apart from glial markers, ENPC are also known to express Nestin and p75NTR. Hence, using a Nestin-GFP mouse, we studied whether ENPC, defined by the co-expression of Nestin and p75NTR, express glial cell markers and whether all enteric glial cells express these ENPC markers. Methods: Cells from the longitudinal muscle-myenteric plexus (LMMP) of Nestin-GFP mice were flow sorted into four populations based on expression of Nestin-GFP and p75NTR for clonal proliferation and differentiation assays. Differentiated cells were tested for the presence of neurons and glia using immunocytochemistry. In separate analyses, these four cell populations were analyzed by FACS for the presence, co-localization, and intensity of fluorescence of glial markers GFAP and S100B. Results: By clonal analyses, we observe that only cells that co-express Nestin and p75NTRHI proliferate (Mean ± S.E. of percentage proliferative cells: 10.2 ± 0.62) and differentiate to form neurons and glia (Mean ± S.E. of percentage of neurospheres that differentiate to neurons and glia: 78.26 ± 4.35). ENPC, defined now by Nestin-p75NTRHI co-expression, express the glial markers, GFAP and S100B. We observe that only 3.86% of enteric glial cells that co-express GFAP and S100B express Nestin-GFP and p75HI and hence qualify for ENPC. All other cells are presumably mature enteric glia. Discussion: We show that ENPC co-express Nestin and p75NTRHI apart from GFAP and S100B, confirming earlier results that ENPC express glial markers. However, ENPC only form a small fraction of the total enteric glial cells as only 3.86% of cells that co-express the glial markers express Nestin-GFP and p75NTRHI. Hence, ENPC either represent a sub-category of enteric glial cells or are non-glial cells expressing shared markers. Our data shows that not all enteric glia are ENPC. This work paves the way for understanding not only the true identity of ENPC, but also that of mature enteric glia.
264 Extracellular Matrix Directs Differentiation of Motor Neuronal Subtypes in Tissue Engineered Innervated Intestinal Smooth Muscle Shreya Raghavan, Khalil N. Bitar Background: Neural stem cell transplantation has the potential to repopulate myenteric ganglia in several functional GI disorders. Cell fate and long term phenotype maintenance are roadblocks when transplanting stem cells into the intestine. We have used extracellular matrix (ECM) microenvironments to direct the differentiation of enteric neural progenitor cells into functional motor neural subtypes in vitro. Objective: The objective of this study was to develop transplantable neural sheets consisting of enriched populations of differentiated motor neurons. Methods: Enteric neural progenitor cells were isolated from jejunal biopsies and cultured to generate enteric neurospheres. ECM microenvironments were designed to include Collagen I, Collagen IV, Laminin and/or heparan sulfate. Rheometry was used to characterize ECM gels. Immunoblotting and immunohistochemistry were used to characterize the presence of differentiated motor neuronal phenotypes. Electrical field stimulation was used to stimulate differentiated neurons, and smooth muscle relaxation was measured to study the presence of inhibitory motor neurons. Results: i) Characterization of ECM gels: Viscoelasticity of ECM gels were within the mechanical range similar to gastrulating mesoderm (0.1-0.5kPa). ii) Neural differentiation within tissue engineered neural sheets: Extent of neural differentiation varied with ECM composition. Neurons on Collagen IV sheets had significant neuronal branching and neural cell clustering. Significantly long neurites (~500μm) were observed in laminin sheets, with evidence of preliminary neuronal networking. "Pre-ganglionic" clusters with inter-neuronal networking were observed in heparan sulfate sheets. iii) Characterization of motor neuronal subtypes: Collagen I sheets had an enriched cholinergic neuronal population, evidenced by a ~1.5 fold increase in choline acetyltransferase expression. Collagen IV sheets had an enriched nitrergic neuron population, with a ~2.5 fold increase in expression of neuronal nitric oxide synthase. iv) Functionality of nitrergic neurons: Stimulation of inhibitory neurons in Collagen IV sheets induced a ~2.7 fold increase in smooth muscle relaxation compared to Collagen I. Magnitude of relaxation in Collagen IV was -413.7±6.7μN compared to -157.3±18.4μN in Collagen I. Pre-treatment with nitric oxide synthase inhibitor attenuated relaxation by 58.9 ± 7.95% in Collagen IV sheets, indicating the presence of nitrergic neurons. Conclusion: We have used ECM composition to direct the differentiation of motor neuronal subtypes within tissue engineered intestinal sheets. This study highlights the potential to generate enriched populations of motor neurons prior to transplantation. Furthermore, differentiated motor neurons were capable of mediating smooth muscle contraction and relaxation. This work was supported by NIH/NIDDK R01 071614. 265 Hedgehog/Gli1 Signaling Regulates Phenotypic Changes in Myeloid Cells During Helicobacter-Induced SPEM (Gastric Metaplasia) Lin Ding, Michael M. Hayes, Amy C. Tsao, Juanita L. Merchant
FACS profiles of GFAP and S100 expression in populations of myenteric plexus cells expressing Nestin and p75NTR
Introduction: Chronic inflammation from Helicobacter infection is a major cause of gastric cancer. We have recently shown that Hedgehog/Gli1 signaling plays an essential role in mediating the pre-neoplastic changes in the stomach called mucous cell metaplasia or SPEM (spasmolytic polypeptide-expressing metaplasia). Gli1-dependent gastric metaplasia coincided with the appearance of Cd11b+Cd11c+Gr1+ myeloid cells, surface markers indicative of myeloid-derived suppressor cells (MDSCs). The myeloid differentiation factor Schlafen4 (Slfn4) is a transcriptional target of Gli1 and is expressed exclusively in this Cd11b+Cd11c+Gr1+ subset of myeloid cells after chronic Helicobacter induction of SPEM. Slfn4 was not expressed in the infected Gli1 null mice. Prior studies have shown that it is strongly induced by type 1 interferons (IFNα11). Aim: To demonstrate that the Gr1+/ Slfn4+ cells identify an MDSC population that is Gli1-dependent. Method: Mice ectopically expressing Sonic hedgehog (pCMV-Shh) and wild type littermates were infected with H. felis for H&E staining and RT-qPCR analysis of corpus. Thioglycolate elicited peritoneal myeloid cells were harvested from WT and Gli1-deficient mice after 96h, cultured overnight then treated with IFNα11. Cd11b+Gr1+ cells were sorted then analyzed for Slfn4 mRNA. Results: Helicobacter-infected pCMV-Shh mice developed a more robust inflammatory response at 3 mos and SPEM by 4 mos after H. felis infection compared to wild type littermates. Thus, sustained Shh expression accelerated the development of SPEM in the infected mice by 2 mos. Slfn4 expression in gastric lysates was 39-fold induced at 4 mos compared to 2-fold at 3 mos after Helicobacter infection and coincided with the appearance of SPEM. IFNα11 (800U/ml) treatment of the cultured peritoneal myeloid cells ex vivo induced a phenotypic shift from Gr1-/Slfn4- to Gr1+/Slfn4+ cells correlating with a 1031fold increase in Slfn4 expression. The expression was Gli1-dependent since there was a 67% decrease in Slfn4 induction if primary mouse myeloid cells were isolated from the Gli1+/mice. In addition, we found that Slfn4 expression was induced >4-fold in the Gr1+ subpopulation compared to the Gr1- cells. Slfn4 induction was 48% or 78% less in the Gr1+ population of the Gli1+/- or Gli1-/- mice respectively. Although loss of Gli1 did not affect the total number of Gr1+ cells, Gli1 was required for IFNα11 Slfn4 induction. Conclusion: Hedgehog ligand over-expression accelerates Helicobacter-induced SPEM and the appearance of Slfn4+ MDSC-like myeloid cells. Slfn4 mRNA was induced mainly in the Gr1+ population in a Gli1-dependent manner. Since IFNα strongly induced Slfn4 and the induction was mitigated by loss of the Gli1 allele, our results suggest cooperative regulation of this myeloid differentiation gene by pro-inflammatory cytokines and Hedgehog signaling.
263 LRIG1 Regulates Differentiation of a Subset of Interstitial Cells of Cajal (ICC) Jumpei Kondo, Anne Powell, Yang Wang, Melissa A. Musser, Michelle Southard-Smith, Robert J. Coffey Background and aims: ICCs serve as pacemakers or neural transmitters that control contraction of smooth muscle to regulate gut motility. ICC dysfunction is thought to be responsible for many human diseases related to gut motility. ICCs are divided into several subpopulations according to their localization: ICC-MY in the myenteric plexus; ICC-IM in circular muscle layer; ICC-DMP and ICC-SMP at inner surface of circular muscle of small intestine and colon, respectively. c-Kit is a well established marker for ICCs and is also important for proper development of ICCs throughout the gastrointestinal tract. c-Kit+ progenitor cells give rise to ICC-MY and intestinal smooth muscle cells during mouse embryogenesis (E12 to E18). The origin of c-Kit-expressing ICC-DMP and ICC-SMP is uncertain; the former can be sparsely observed at birth in murine jejunum and the latter at postnatal day 5 (P5) in proximal colon. Here, we show that ICC-DMP and ICC-SMP express Lrig1, a recently identified intestinal epithelial stem cell marker. Methods: Wild-type and Lrig1-null mice were used for immunohistochemical experiments and intestinal motility assays. Intestinal motility was evaluated by passage of Rhodamine-B conjugated dextran in 5% methylcellulose, which was given orally. Lrig1-Apple reporter mice, in which exon 1 of Lrig1 gene is replaced by a sequence encoding apple fluorescent protein, were used to confirm Lrig1 expression in ICC-DMP and ICC-SMP. For lineage tracing, Lrig1-CreERT2; Rosa26R-YFP mice were given a single intraperitoneal dose of 33mg/kg tamoxifen. Mice were sacrificed at P2 to P15, and YFP expression in small intestine and colon was evaluated by immunohistochemistry. Results: Immunohistochemical study of wild-type mice revealed that ICC-DMP and ICCSMP express Lrig1. Mice lacking Lrig1 protein show loss of c-Kit staining in the DMP and SMP region, as well as absent staining for the ICC markers, Ano1 and NK1R, in that area. Of interest, Lrig1-null mice exhibit slower intestinal motility. In newborn mice, Lrig1 is expressed by smooth muscle cells and exhibits a clear gradient with strong expression at the inner border of circular muscle layer and weak expression at the outer border. At P5,
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AGA Abstracts
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Identification and Characterization of a Subset of Enteric "Glial" Cells As Neural Precursors Subhash Kulkarni, Cuiping Li, Maria-Adelaide Micci, Pankaj J. Pasricha Introduction: Recently, it has been shown that cells expressing the glial marker GFAP can make enteric neurons, raising the possibility that enteric glia can function as enteric neural precursor cells (ENPC). However, it is not known whether all or only a sub-population of enteric glia are ENPC. Apart from glial markers, ENPC are also known to express Nestin and p75NTR. Hence, using a Nestin-GFP mouse, we studied whether ENPC, defined by the co-expression of Nestin and p75NTR, express glial cell markers and whether all enteric glial cells express these ENPC markers. Methods: Cells from the longitudinal muscle-myenteric plexus (LMMP) of Nestin-GFP mice were flow sorted into four populations based on expression of Nestin-GFP and p75NTR for clonal proliferation and differentiation assays. Differentiated cells were tested for the presence of neurons and glia using immunocytochemistry. In separate analyses, these four cell populations were analyzed by FACS for the presence, co-localization, and intensity of fluorescence of glial markers GFAP and S100B. Results: By clonal analyses, we observe that only cells that co-express Nestin and p75NTRHI proliferate (Mean ± S.E. of percentage proliferative cells: 10.2 ± 0.62) and differentiate to form neurons and glia (Mean ± S.E. of percentage of neurospheres that differentiate to neurons and glia: 78.26 ± 4.35). ENPC, defined now by Nestin-p75NTRHI co-expression, express the glial markers, GFAP and S100B. We observe that only 3.86% of enteric glial cells that co-express GFAP and S100B express Nestin-GFP and p75HI and hence qualify for ENPC. All other cells are presumably mature enteric glia. Discussion: We show that ENPC co-express Nestin and p75NTRHI apart from GFAP and S100B, confirming earlier results that ENPC express glial markers. However, ENPC only form a small fraction of the total enteric glial cells as only 3.86% of cells that co-express the glial markers express Nestin-GFP and p75NTRHI. Hence, ENPC either represent a sub-category of enteric glial cells or are non-glial cells expressing shared markers. Our data shows that not all enteric glia are ENPC. This work paves the way for understanding not only the true identity of ENPC, but also that of mature enteric glia.
264 Extracellular Matrix Directs Differentiation of Motor Neuronal Subtypes in Tissue Engineered Innervated Intestinal Smooth Muscle Shreya Raghavan, Khalil N. Bitar Background: Neural stem cell transplantation has the potential to repopulate myenteric ganglia in several functional GI disorders. Cell fate and long term phenotype maintenance are roadblocks when transplanting stem cells into the intestine. We have used extracellular matrix (ECM) microenvironments to direct the differentiation of enteric neural progenitor cells into functional motor neural subtypes in vitro. Objective: The objective of this study was to develop transplantable neural sheets consisting of enriched populations of differentiated motor neurons. Methods: Enteric neural progenitor cells were isolated from jejunal biopsies and cultured to generate enteric neurospheres. ECM microenvironments were designed to include Collagen I, Collagen IV, Laminin and/or heparan sulfate. Rheometry was used to characterize ECM gels. Immunoblotting and immunohistochemistry were used to characterize the presence of differentiated motor neuronal phenotypes. Electrical field stimulation was used to stimulate differentiated neurons, and smooth muscle relaxation was measured to study the presence of inhibitory motor neurons. Results: i) Characterization of ECM gels: Viscoelasticity of ECM gels were within the mechanical range similar to gastrulating mesoderm (0.1-0.5kPa). ii) Neural differentiation within tissue engineered neural sheets: Extent of neural differentiation varied with ECM composition. Neurons on Collagen IV sheets had significant neuronal branching and neural cell clustering. Significantly long neurites (~500μm) were observed in laminin sheets, with evidence of preliminary neuronal networking. "Pre-ganglionic" clusters with inter-neuronal networking were observed in heparan sulfate sheets. iii) Characterization of motor neuronal subtypes: Collagen I sheets had an enriched cholinergic neuronal population, evidenced by a ~1.5 fold increase in choline acetyltransferase expression. Collagen IV sheets had an enriched nitrergic neuron population, with a ~2.5 fold increase in expression of neuronal nitric oxide synthase. iv) Functionality of nitrergic neurons: Stimulation of inhibitory neurons in Collagen IV sheets induced a ~2.7 fold increase in smooth muscle relaxation compared to Collagen I. Magnitude of relaxation in Collagen IV was -413.7±6.7μN compared to -157.3±18.4μN in Collagen I. Pre-treatment with nitric oxide synthase inhibitor attenuated relaxation by 58.9 ± 7.95% in Collagen IV sheets, indicating the presence of nitrergic neurons. Conclusion: We have used ECM composition to direct the differentiation of motor neuronal subtypes within tissue engineered intestinal sheets. This study highlights the potential to generate enriched populations of motor neurons prior to transplantation. Furthermore, differentiated motor neurons were capable of mediating smooth muscle contraction and relaxation. This work was supported by NIH/NIDDK R01 071614. 265 Hedgehog/Gli1 Signaling Regulates Phenotypic Changes in Myeloid Cells During Helicobacter-Induced SPEM (Gastric Metaplasia) Lin Ding, Michael M. Hayes, Amy C. Tsao, Juanita L. Merchant
FACS profiles of GFAP and S100 expression in populations of myenteric plexus cells expressing Nestin and p75NTR
Introduction: Chronic inflammation from Helicobacter infection is a major cause of gastric cancer. We have recently shown that Hedgehog/Gli1 signaling plays an essential role in mediating the pre-neoplastic changes in the stomach called mucous cell metaplasia or SPEM (spasmolytic polypeptide-expressing metaplasia). Gli1-dependent gastric metaplasia coincided with the appearance of Cd11b+Cd11c+Gr1+ myeloid cells, surface markers indicative of myeloid-derived suppressor cells (MDSCs). The myeloid differentiation factor Schlafen4 (Slfn4) is a transcriptional target of Gli1 and is expressed exclusively in this Cd11b+Cd11c+Gr1+ subset of myeloid cells after chronic Helicobacter induction of SPEM. Slfn4 was not expressed in the infected Gli1 null mice. Prior studies have shown that it is strongly induced by type 1 interferons (IFNα11). Aim: To demonstrate that the Gr1+/ Slfn4+ cells identify an MDSC population that is Gli1-dependent. Method: Mice ectopically expressing Sonic hedgehog (pCMV-Shh) and wild type littermates were infected with H. felis for H&E staining and RT-qPCR analysis of corpus. Thioglycolate elicited peritoneal myeloid cells were harvested from WT and Gli1-deficient mice after 96h, cultured overnight then treated with IFNα11. Cd11b+Gr1+ cells were sorted then analyzed for Slfn4 mRNA. Results: Helicobacter-infected pCMV-Shh mice developed a more robust inflammatory response at 3 mos and SPEM by 4 mos after H. felis infection compared to wild type littermates. Thus, sustained Shh expression accelerated the development of SPEM in the infected mice by 2 mos. Slfn4 expression in gastric lysates was 39-fold induced at 4 mos compared to 2-fold at 3 mos after Helicobacter infection and coincided with the appearance of SPEM. IFNα11 (800U/ml) treatment of the cultured peritoneal myeloid cells ex vivo induced a phenotypic shift from Gr1-/Slfn4- to Gr1+/Slfn4+ cells correlating with a 1031fold increase in Slfn4 expression. The expression was Gli1-dependent since there was a 67% decrease in Slfn4 induction if primary mouse myeloid cells were isolated from the Gli1+/mice. In addition, we found that Slfn4 expression was induced >4-fold in the Gr1+ subpopulation compared to the Gr1- cells. Slfn4 induction was 48% or 78% less in the Gr1+ population of the Gli1+/- or Gli1-/- mice respectively. Although loss of Gli1 did not affect the total number of Gr1+ cells, Gli1 was required for IFNα11 Slfn4 induction. Conclusion: Hedgehog ligand over-expression accelerates Helicobacter-induced SPEM and the appearance of Slfn4+ MDSC-like myeloid cells. Slfn4 mRNA was induced mainly in the Gr1+ population in a Gli1-dependent manner. Since IFNα strongly induced Slfn4 and the induction was mitigated by loss of the Gli1 allele, our results suggest cooperative regulation of this myeloid differentiation gene by pro-inflammatory cytokines and Hedgehog signaling.
263 LRIG1 Regulates Differentiation of a Subset of Interstitial Cells of Cajal (ICC) Jumpei Kondo, Anne Powell, Yang Wang, Melissa A. Musser, Michelle Southard-Smith, Robert J. Coffey Background and aims: ICCs serve as pacemakers or neural transmitters that control contraction of smooth muscle to regulate gut motility. ICC dysfunction is thought to be responsible for many human diseases related to gut motility. ICCs are divided into several subpopulations according to their localization: ICC-MY in the myenteric plexus; ICC-IM in circular muscle layer; ICC-DMP and ICC-SMP at inner surface of circular muscle of small intestine and colon, respectively. c-Kit is a well established marker for ICCs and is also important for proper development of ICCs throughout the gastrointestinal tract. c-Kit+ progenitor cells give rise to ICC-MY and intestinal smooth muscle cells during mouse embryogenesis (E12 to E18). The origin of c-Kit-expressing ICC-DMP and ICC-SMP is uncertain; the former can be sparsely observed at birth in murine jejunum and the latter at postnatal day 5 (P5) in proximal colon. Here, we show that ICC-DMP and ICC-SMP express Lrig1, a recently identified intestinal epithelial stem cell marker. Methods: Wild-type and Lrig1-null mice were used for immunohistochemical experiments and intestinal motility assays. Intestinal motility was evaluated by passage of Rhodamine-B conjugated dextran in 5% methylcellulose, which was given orally. Lrig1-Apple reporter mice, in which exon 1 of Lrig1 gene is replaced by a sequence encoding apple fluorescent protein, were used to confirm Lrig1 expression in ICC-DMP and ICC-SMP. For lineage tracing, Lrig1-CreERT2; Rosa26R-YFP mice were given a single intraperitoneal dose of 33mg/kg tamoxifen. Mice were sacrificed at P2 to P15, and YFP expression in small intestine and colon was evaluated by immunohistochemistry. Results: Immunohistochemical study of wild-type mice revealed that ICC-DMP and ICCSMP express Lrig1. Mice lacking Lrig1 protein show loss of c-Kit staining in the DMP and SMP region, as well as absent staining for the ICC markers, Ano1 and NK1R, in that area. Of interest, Lrig1-null mice exhibit slower intestinal motility. In newborn mice, Lrig1 is expressed by smooth muscle cells and exhibits a clear gradient with strong expression at the inner border of circular muscle layer and weak expression at the outer border. At P5,
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AGA Abstracts
262