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Spatial and Temporal Differences Between Enteric Neural Progenitors From Small Intestine of Adult Mice
AGA Abstracts
Su1967
Su1968
ROCK1/2 Inhibitor Induces Enteric Phenotype in CNS Derived Neural Stem Cells Gunjan Tiwari, Jung Hwan Oh, Laren Becker, Subhash Kulkarni, Johann Peterson, MariaAdelaide Micci, Pankaj J. Pasricha
Spatial and Temporal Differences Between Enteric Neural Progenitors From Small Intestine of Adult Mice Laren Becker, Subhash Kulkarni, Gunjan Tiwari, Maria-Adelaide Micci, Johann Peterson, Pankaj J. Pasricha
Introduction Central nervous system-derived neural stem cells (CNS-NSC) have been previously shown to populate the enteric nervous system after transplantation in the gut. CNSNSC, are therefore considered promising candidates for cell based therapy for enteric neuropathies such as achalasia and Hirschsprung's disease. However the optimal conditions leading to the most efficient conversion of CNS-NSC to an enteric phenotype are still being investigated. Here we describe a simple and efficient method of inducing enteric phenotype in CNSNSC using the ROCK1/2 inhibitor Y27632. Methods CNS-NSC isolated from the subventricular zone of E13.5 mouse embryos were cultured under proliferating conditions in presence or absence of 25uM Y27632 and the expression of enteric markers was examined by qRT-PCR after 5 days. Results The CNS-NSC cultured in presence of Y27632 formed neurospheres that adhered and cells started migrating out of them and growing neural processes. On the other hand, CNS-NSC cultured in the absence of Y27632 continued to proliferate and grow as floating neurospheres. qRT-PCR analysis of cells cultured with Y27632 revealed 2.5-fold increase in the expression of p75, an enteric neural progenitor marker and 4.5 fold increase in the expression of Ret, a key regulator of enteric neuronal development over cells cultured without Y27632. The expression of Sox 10, a glial cell marker, was however found to be 3.3 fold lower in cells cultured with Y27632, suggesting preferential differentiation of CNS-NSC into neurons in presence of Y27632. Conclusions Inhibition of ROCK1/2 is a novel, highly efficient and less manipulative strategy for inducing enteric phenotype in NSC derived from CNS and it can potentially be expanded to induce enteric phenotype on stem cells of diverse origin.
Introduction: The discovery of enteric neural progenitors (ENPs) in adult rodent and human gut has tremendous implications with respect to disease pathogenesis and regenerative medicine. However, little is known on whether and how ENPs differ with age of the individual and the location in different regions of the gut. We hypothesize that such spatial and temporal variables will affect ENP behavior. Method: To study the influence of spatial orientation on ENP behavior, small intestine from C57BL/6 and Nestin-GFP (Nes-Gfp) mice (age 2-4 months) was dissected into two layers: the outer layer containing the longitudinal muscle and myenteric plexus (LM-MP) and the inner layer containing the circular muscle, submucosal plexus and mucosa (SMP). ENPs, isolated from digested LM-MP and SMP, were grown as neurospheres and then differentiated (by a withdrawl of growth factors) on fibronectincoated coverslips. Nes-Gfp+ cells were sorted from by FACS, and their RNA was isolated immediately post-sort and on differentiation. To examine the temporal influence on stem cell behavior, ENPs were isolated from the LM-MP layer of two groups of C57BL/6 mice: aged (25-29 months) and young (2-3 months). Isolated cells were grown to neurospheres and differentiated. Cells were examined before and after differentiation by immunocytochemistry and qRT-PCR. Results: ENPs isolated from LM-MP preferentially differentiated into neurons and glial cells, as evidenced by immunostaining and increased mRNA expression of βIII-tubulin (2-fold), ChAT (144-fold), NOS1 (66-fold), VIP (203-fold), and GFAP (748fold), compared to SMP, while those from the SMP layer formed more smooth muscle cells with an 11-fold increase in mRNA expression of ACTG2 (smooth muscle actin) compared to LM-MP. The proliferating neurospheres from LM-MP expressed substantially higher levels of neural crest markers p75 (157-fold) and Sox10 (142-fold), and neural progenitor marker, Nestin (3-fold). The same expression pattern was also present after sorting for Nes-Gfp+ cells from the two layers, suggesting intrinsic differences in the stem cells rather than heterogeneity explains the observed phenotye. Upon differentiation, ENPs from aged mice were capable of differentiating into neurons, glial cells and smooth muscle cells, but formed less cholinergic neurons (3-fold less ChAT expression) than those from young mice. Conclusion: We find that spatial and temporal variables do affect ENP behavior. Su1969 Gene Expression Analysis of the Enteric Nervous System in Germ Free Mice Subhash Kulkarni, Laren Becker, Gunjan Tiwari, Johann Peterson, Maria-Adelaide Micci, Justin Sonnenburg, Pankaj J. Pasricha Background: The absence of gut microbiota, resident largely in the cecum and colon of rodents, is known to increase gut transit times in germ free (GF) animals, when compared to conventional (Conv) animals. Although the enteric nervous system (ENS) is known to regulate gut motility, the effect of the absence of gut microbiota on distribution and gene expression of specific compartments of the ENS has not yet been studied. Methods:: Age, gender and chow matched GF and Conv mice (five each) were weighed and then sacrificed and their gut contents were flushed out. Small intestine, cecum and colon was dissected out and their lengths were measured. The colon muscle layer (CML) and total cecum was flash frozen for total RNA extraction. Whole genome expression differences between RNA pooled from individual germ free and conventional cecum and CML were characterized using microarray analysis. Pathway analysis was performed on differentially expressed genes. Results: We found a significant difference between the groups in their weight (GF: 38.4 ± 1.08 g, Conv: 44 ± 1.05g; p = 0.006) and in the length of cecum (GF: 4.2 ± 0.25 cm, Conv: 2.28 ± 0.16 cm; p < 0.0001). Pathway analysis on differentially expressed genes identified nervous system development and function, both in the cecum and the CML, between GF and Conv animals. In the cecum, we found a 3.45 fold over-expression of PYY in GF animals, known to lower motility in the gut through neuropeptide Y neurons and lower appetite through POMC neurons, when compared to Conv animals. In the CML, we found an increase in expression of genes such as PGP9.5 (Uchl1; 2.81 fold), Gremlin (Grem1; 4.18 fold), GDNF (2.15 fold), Foxf2 (2.26 fold), VIP (2.10 fold) and Smoothelin (Smtn; 4.05 fold) in GF animals when compared to Conv animals. Conclusion: Our data suggests significant differences between GF and Conv animals with respect to patterns of enteric neural and muscle development. These findings have significant implications for the understanding of the enteric neuromuscular responses to alterations in the gut microbiota in various motility disorders.
CNS-NSC cultured without Y27632
Su1970 Electroacupuncture With High Frequency at Acupoint ST-36 Induces Regeneration of Lost Enteric Neurons in Diabetic Rats via GDNF and PI3K/ AKT Signal Pathway Fan Du, Shi Liu Background: Electroacupuncture (EA) at acupoint ST-36 (Zusanli) has been used to alleviate gastrointestinal symptoms, improve gastrointestinal motility and treat functional gastrointestinal diseases. Our previous study showed that diabetes can significantly induce enteric neuronal loss. Glial cell line-derived neurotrophic factor (GDNF) and its downstream signal pathway PI3K/Akt may play an important role in enteric neuropathy in diabetes. But the effects and mechanisms of EA on enteric nervous system (ENS) have scarcely been investigated. Aim: To investigate the effects of EA at acupoint ST-36 on ENS and to explore the possible mechanism. Methods: 40 SD male rats were randomly divided into five groups (every subgroup N=8): normal control group, diabetic group (DM), diabetic with high frequency EA group (HEA), diabetic with low frequency EA group (LEA) and diabetic with sham EA group (SEA). Diabetes was induced by a single intraperitoneal injection of Streptozotocin (STZ) at 60 mg/kg. The parameters of HEA included a frequency of 100HZ and an amplitude of 1mA, while the LEA were 10HZ and 1mA; the SEA was that needles were inserted into the acupoints but without current. The three EA groups were given true or sham EA 30
CNS-NSC cultured with Y27632
AGA Abstracts
S-522
Su1967
Su1968
ROCK1/2 Inhibitor Induces Enteric Phenotype in CNS Derived Neural Stem Cells Gunjan Tiwari, Jung Hwan Oh, Laren Becker, Subhash Kulkarni, Johann Peterson, MariaAdelaide Micci, Pankaj J. Pasricha
Spatial and Temporal Differences Between Enteric Neural Progenitors From Small Intestine of Adult Mice Laren Becker, Subhash Kulkarni, Gunjan Tiwari, Maria-Adelaide Micci, Johann Peterson, Pankaj J. Pasricha
Introduction Central nervous system-derived neural stem cells (CNS-NSC) have been previously shown to populate the enteric nervous system after transplantation in the gut. CNSNSC, are therefore considered promising candidates for cell based therapy for enteric neuropathies such as achalasia and Hirschsprung's disease. However the optimal conditions leading to the most efficient conversion of CNS-NSC to an enteric phenotype are still being investigated. Here we describe a simple and efficient method of inducing enteric phenotype in CNSNSC using the ROCK1/2 inhibitor Y27632. Methods CNS-NSC isolated from the subventricular zone of E13.5 mouse embryos were cultured under proliferating conditions in presence or absence of 25uM Y27632 and the expression of enteric markers was examined by qRT-PCR after 5 days. Results The CNS-NSC cultured in presence of Y27632 formed neurospheres that adhered and cells started migrating out of them and growing neural processes. On the other hand, CNS-NSC cultured in the absence of Y27632 continued to proliferate and grow as floating neurospheres. qRT-PCR analysis of cells cultured with Y27632 revealed 2.5-fold increase in the expression of p75, an enteric neural progenitor marker and 4.5 fold increase in the expression of Ret, a key regulator of enteric neuronal development over cells cultured without Y27632. The expression of Sox 10, a glial cell marker, was however found to be 3.3 fold lower in cells cultured with Y27632, suggesting preferential differentiation of CNS-NSC into neurons in presence of Y27632. Conclusions Inhibition of ROCK1/2 is a novel, highly efficient and less manipulative strategy for inducing enteric phenotype in NSC derived from CNS and it can potentially be expanded to induce enteric phenotype on stem cells of diverse origin.
Introduction: The discovery of enteric neural progenitors (ENPs) in adult rodent and human gut has tremendous implications with respect to disease pathogenesis and regenerative medicine. However, little is known on whether and how ENPs differ with age of the individual and the location in different regions of the gut. We hypothesize that such spatial and temporal variables will affect ENP behavior. Method: To study the influence of spatial orientation on ENP behavior, small intestine from C57BL/6 and Nestin-GFP (Nes-Gfp) mice (age 2-4 months) was dissected into two layers: the outer layer containing the longitudinal muscle and myenteric plexus (LM-MP) and the inner layer containing the circular muscle, submucosal plexus and mucosa (SMP). ENPs, isolated from digested LM-MP and SMP, were grown as neurospheres and then differentiated (by a withdrawl of growth factors) on fibronectincoated coverslips. Nes-Gfp+ cells were sorted from by FACS, and their RNA was isolated immediately post-sort and on differentiation. To examine the temporal influence on stem cell behavior, ENPs were isolated from the LM-MP layer of two groups of C57BL/6 mice: aged (25-29 months) and young (2-3 months). Isolated cells were grown to neurospheres and differentiated. Cells were examined before and after differentiation by immunocytochemistry and qRT-PCR. Results: ENPs isolated from LM-MP preferentially differentiated into neurons and glial cells, as evidenced by immunostaining and increased mRNA expression of βIII-tubulin (2-fold), ChAT (144-fold), NOS1 (66-fold), VIP (203-fold), and GFAP (748fold), compared to SMP, while those from the SMP layer formed more smooth muscle cells with an 11-fold increase in mRNA expression of ACTG2 (smooth muscle actin) compared to LM-MP. The proliferating neurospheres from LM-MP expressed substantially higher levels of neural crest markers p75 (157-fold) and Sox10 (142-fold), and neural progenitor marker, Nestin (3-fold). The same expression pattern was also present after sorting for Nes-Gfp+ cells from the two layers, suggesting intrinsic differences in the stem cells rather than heterogeneity explains the observed phenotye. Upon differentiation, ENPs from aged mice were capable of differentiating into neurons, glial cells and smooth muscle cells, but formed less cholinergic neurons (3-fold less ChAT expression) than those from young mice. Conclusion: We find that spatial and temporal variables do affect ENP behavior. Su1969 Gene Expression Analysis of the Enteric Nervous System in Germ Free Mice Subhash Kulkarni, Laren Becker, Gunjan Tiwari, Johann Peterson, Maria-Adelaide Micci, Justin Sonnenburg, Pankaj J. Pasricha Background: The absence of gut microbiota, resident largely in the cecum and colon of rodents, is known to increase gut transit times in germ free (GF) animals, when compared to conventional (Conv) animals. Although the enteric nervous system (ENS) is known to regulate gut motility, the effect of the absence of gut microbiota on distribution and gene expression of specific compartments of the ENS has not yet been studied. Methods:: Age, gender and chow matched GF and Conv mice (five each) were weighed and then sacrificed and their gut contents were flushed out. Small intestine, cecum and colon was dissected out and their lengths were measured. The colon muscle layer (CML) and total cecum was flash frozen for total RNA extraction. Whole genome expression differences between RNA pooled from individual germ free and conventional cecum and CML were characterized using microarray analysis. Pathway analysis was performed on differentially expressed genes. Results: We found a significant difference between the groups in their weight (GF: 38.4 ± 1.08 g, Conv: 44 ± 1.05g; p = 0.006) and in the length of cecum (GF: 4.2 ± 0.25 cm, Conv: 2.28 ± 0.16 cm; p < 0.0001). Pathway analysis on differentially expressed genes identified nervous system development and function, both in the cecum and the CML, between GF and Conv animals. In the cecum, we found a 3.45 fold over-expression of PYY in GF animals, known to lower motility in the gut through neuropeptide Y neurons and lower appetite through POMC neurons, when compared to Conv animals. In the CML, we found an increase in expression of genes such as PGP9.5 (Uchl1; 2.81 fold), Gremlin (Grem1; 4.18 fold), GDNF (2.15 fold), Foxf2 (2.26 fold), VIP (2.10 fold) and Smoothelin (Smtn; 4.05 fold) in GF animals when compared to Conv animals. Conclusion: Our data suggests significant differences between GF and Conv animals with respect to patterns of enteric neural and muscle development. These findings have significant implications for the understanding of the enteric neuromuscular responses to alterations in the gut microbiota in various motility disorders.
CNS-NSC cultured without Y27632
Su1970 Electroacupuncture With High Frequency at Acupoint ST-36 Induces Regeneration of Lost Enteric Neurons in Diabetic Rats via GDNF and PI3K/ AKT Signal Pathway Fan Du, Shi Liu Background: Electroacupuncture (EA) at acupoint ST-36 (Zusanli) has been used to alleviate gastrointestinal symptoms, improve gastrointestinal motility and treat functional gastrointestinal diseases. Our previous study showed that diabetes can significantly induce enteric neuronal loss. Glial cell line-derived neurotrophic factor (GDNF) and its downstream signal pathway PI3K/Akt may play an important role in enteric neuropathy in diabetes. But the effects and mechanisms of EA on enteric nervous system (ENS) have scarcely been investigated. Aim: To investigate the effects of EA at acupoint ST-36 on ENS and to explore the possible mechanism. Methods: 40 SD male rats were randomly divided into five groups (every subgroup N=8): normal control group, diabetic group (DM), diabetic with high frequency EA group (HEA), diabetic with low frequency EA group (LEA) and diabetic with sham EA group (SEA). Diabetes was induced by a single intraperitoneal injection of Streptozotocin (STZ) at 60 mg/kg. The parameters of HEA included a frequency of 100HZ and an amplitude of 1mA, while the LEA were 10HZ and 1mA; the SEA was that needles were inserted into the acupoints but without current. The three EA groups were given true or sham EA 30
CNS-NSC cultured with Y27632
AGA Abstracts
S-522