Gastroenterology
Volume 129, Issue 6 , Pages 1817-1824, December 2005

Neural Stem Cell Transplantation in the Stomach Rescues Gastric Function in Neuronal Nitric Oxide Synthase–Deficient Mice

Enteric Neuromuscular Disorders and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston, Texas

Received 22 June 2005; accepted 24 August 2005. published online 04 October 2005.

Article Outline

Background & Aims: Nitric oxide is a major inhibitory neurotransmitter in the enteric nervous system. Loss or dysfunction of nitrinergic neurons is associated with serious disruptions of motility, intractable symptoms, and long-term suffering. The aim of this study was to evaluate the effect of intrapyloric transplantation of neural stem cells (NSCs) on gastric emptying and pyloric function in nNOS−/− mice, a well-established genetic model of gastroparesis. Methods: NSCs were isolated from embryonic mice transgenically engineered to express green fluorescent protein and transplanted into the pylorus of nNOS−/− mice. Grafted cells were visualized in pyloric sections and further characterized by immunofluorescence staining. One week posttransplantation, gastric emptying to a non-nutrient meal was measured using the phenol red method and pyloric function was assessed by measuring the relaxation of pyloric strips in an organ bath in response to electrical field stimulation (EFS) under nonadrenergic, noncholinergic conditions. Results: One week following implantation, grafted NSCs differentiated into neurons and expressed neuronal nitric oxide synthase. Gastric emptying was significantly increased in mice that received NSCs as compared with vehicle-injected controls (49.67% vs 35.09%; P < .01 by Student t test). EFS-induced relaxation of pyloric strips was also significantly increased (P < .01 by 2-way analysis of variance). The nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester and the neuronal blocker tetrodotoxin blocked the EFS-induced relaxation, indicating that the observed effect is NO mediated and neuronally derived. Conclusions: Our results support the potential of NSC transplantation as a viable therapeutic option for neuroenteric disorders.

Abbreviations used in this paper:  CNS, central nervous system , EFS, electrical field stimulation , GFAP, glial fibrillary acidic protein , GFP, green fluorescent protein , l-NAME, NG-nitro-l-arginine methyl ester , NANC, nonadrenergicnoncholinergic , nNOS, neuronal nitric oxide synthase , NSC, neural stem cell , PGP9.5, protein gene product 9.5 , TTX, tetrodotoxin , VIP, vasoactive intestinal polypeptide

 

The enteric nervous system is a well-defined system of neurons that regulates several aspects of gastrointestinal physiology, including motility and secretion.1 Normal bolus propagation down the alimentary tract is dependent both on cephalad excitation of gut segments producing propulsive pressure and on caudad relaxation and reduction in flow resistance. Further, active relaxation of gastrointestinal sphincters is critical to prevent functional obstruction at these regions. Motor neurons of the myenteric plexus, which can be either excitatory or inhibitory in nature, are responsible for the immediate neural control of gut muscle tone. The major excitatory motor pathway involves acetylcholine and tachykinins such as substance P and neurokinin A, whereas the main inhibitory neurotransmitters are nitric oxide (NO), vasoactive intestinal polypeptide (VIP), and adenosine triphosphate. Of these, NO, produced by the enzyme neuronal nitric oxide synthase (nNOS), plays a critical role.2

As shown by pharmacological studies and by nNOS-deficient mice, NO deficiency produces nearly complete loss of nerve-stimulated smooth muscle relaxation in the lower esophageal sphincter and pyloric sphincter with less striking changes in the small and large intestines.3, 4, 5 However, the relative sparing of these organs in this model may reflect the expression of alternative splice isoforms of nNOS and not necessarily signify a diminution of the role of NO in these regions.6, 7 By contrast, the stomach of nNOS−/− mice is strikingly dilated with an associated marked delay in emptying for both solids and liquids, reflecting a generalized neuromuscular defect affecting both the pyloric sphincter and the body of the stomach.4

Evidence also exists that loss or dysfunction of enteric nervous system inhibitory neurons plays a role in diabetic gastroparesis, achalasia, and intestinal pseudo-obstruction, clinical disorders associated with serious disruptions of motility, intractable symptoms, and long-term suffering.8, 9, 10 The lack of effective pharmacological or other conventional therapies for these syndromes has led us to explore the possibilities of addressing the underlying neurotransmitter deficiency using novel approaches such as neural stem cell (NSC) transplantation. NSCs are immature, uncommitted cells with the ability to self-renew and to give rise to an array of specialized cells, including neurons.11, 12 Following in vivo implantation in the brain and other structures, NSCs have been shown to produce phenotypes that are characteristic and appropriate for the environment into which they are transplanted, without disrupting the normal dynamics of the target tissue.13, 14 We demonstrate here that intrapyloric transplantation of NSCs can restore nitrinergic neurotransmission and significantly improve gastric function in nNOS−/− transgenic mice, a well-established genetic model of gastroparesis.

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Materials and Methods 

Animals 

Staged-pregnant female green fluorescent protein (GFP) mice (TgN[GFPU]5Nagy; Jackson Laboratories, Bar Harbor, ME) at embryonic day 15 were used for the isolation of NSCs. Adult male nNOS−/− mice (B6;129S4-NOS1Tm1h/J; Jackson Laboratories) were used in all transplantation experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch (Galveston, TX) in accordance with the guidelines provided by the National Institutes of Health.

Isolation and In Vitro Culture of Mouse NSCs 

Cell culture reagents were obtained from Invitrogen (Carlsbad, CA) except where noted. Staged-pregnant female GFP mice were anesthetized with sodium pentobarbital (70 mg/kg intraperitoneally), and a midline incision was made to expose the embryos. The brains of embryonic mice were removed and the subventricular zone dissected from each brain hemisphere. The tissues were washed in Ca2+- and Mg2+-free Hank’s buffered salt solution and digested using a combination of dispase type I (0.1%) and trypsin (0.005%) for 10 minutes at 37°C. After digestion, a cell suspension was obtained by gentle trituration, pelleted, and resuspended in neurobasal medium containing B27, 2 mmol/L glutamine and penicillin-streptomycin (NB27), plus 20 ng/mL fibroblast growth factor and 20 ng/mL epidermal growth factor (Promega, Madison, WI). Under these conditions, embryonic NSCs can be propagated in culture for several weeks, retaining their undifferentiated state.

Recipient Animals and Transplantation 

For transplantation, isolated GFP-NSCs were suspended at a concentration of 50,000 cells/μL in Dulbecco’s phosphate-buffered saline (PBS) containing the caspase-1 inhibitor Ac-YVAD-cmk (500 μmol/L; Calbiochem, La Jolla, CA) and kept on ice. Experimental recipient mice (6–8 weeks old) were deeply anesthetized with sodium pentobarbital (70 mg/kg intraperitoneally). A midabdominal incision was made and the pylorus identified. Two microliters of NSC suspension was injected bilaterally into the midpylorus immediately subserosal using a 22-gauge needle attached to a 10-μL Hamilton syringe.

Tissue Processing and Immunohistochemical Analysis of Grafted NSCs 

One week following NSC transplantation, mice were anesthetized with sodium pentobarbital (70 mg/kg intraperitoneally), transcardially perfused, and fixed with freshly prepared ice-cold 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4). The pylorus was removed, postfixed in 4% paraformaldehyde, and cryoprotected by infiltration in 20% sucrose solution in PBS overnight at 4°C. The tissue was rapidly frozen in O.C.T. embedding medium (Tissue Tek; Sakura, Tokyo, Japan) over dry ice–chilled isopentane (Sigma Chemical Co, St Louis, MO). Frozen serial sections (12 μm thick) were cut on a cryostat (TBS, Durham, NC), placed on gelatin-coated slides (VWR, West Chester, PA), and stored at −80°C until needed. Frozen sections were blocked and permeabilized for 1 hour at room temperature with 0.3% Triton X-100 in PBS containing 5% normal goat serum. After washing in PBS, sections were incubated with primary antibodies diluted in PBS containing 1.5% normal goat serum overnight at 4°C. The following antibodies were used: rabbit anti–protein gene product 9.5 (PGP9.5, 1:2000; Chemicon, Temecula, CA), rabbit anti–glial fibrillary acidic protein (GFAP, 1:1500; Chemicon), rabbit anti-nNOS (1:200; Zymed, San Francisco, CA), and rabbit anti-VIP (1:100; Alpha Diagnostic International, Inc, San Antonio, TX). After washing, sections were incubated for 1 hour at room temperature with Alexa-conjugated secondary antibodies (Alexa-594, 1:400 dilution; Molecular Probes, Eugene, OR). After 2 more washes, sections were cover-slipped with Fluorsave mounting medium (Calbiochem, La Jolla, CA). Staining controls were produced by omitting the primary antibodies.

Microscope and Imaging System 

Sections were examined with a confocal laser scanning microscope (LSM510 META; Carl Zeiss Inc, Thornwood, NY) equipped with 3 lasers for the multiple detection of several emission signals with complete separation of their wavelengths.

Gastric Emptying 

Mice were fasted 18 hours and were allowed free access to water. Gastric emptying of liquids was assessed by gavage of a 0.3-mL solution of 0.05% phenol red (Sigma Chemical Co) administered 20 minutes before killing each mouse. Three additional mice were also killed immediately after intragastric administration of phenol red for baseline control. The stomach of each mouse was removed after ligation of both the cardiac and pyloric ends and placed in 10 mL of 0.1N NaOH. The stomach and its contents were homogenized for 30 seconds at medium speed, and the mixture was kept for 1 hour at room temperature. Phenol red content was measured according to the method by Scarpignato et al15 and as previously described by Mashimo et al.4 Briefly, 0.5 mL of supernatant was added to 0.05 mL of 20% acetoacetic acid. After centrifugation at 2500g for 20 minutes, 0.4 mL of 0.5N NaOH was added to the supernatant. The absorbance of the sample was read at 560 nm by a spectrophotometer. Gastric emptying (GE) for each mouse was calculated using the following formula:

Organ Bath Physiology 

Mice were killed by cervical dislocation and the stomach removed and placed in Krebs buffer. The pyloric region was dissected, and circular muscle strips were mounted between 2 L-shaped tissue hooks in 5-mL chambers containing Krebs buffer at 37°C and continuously bubbled with 95% o2/5% co2 (Radnoti Glass Technology, Monrovia, CA). Tension was monitored with an isometric force transducer and recorded and analyzed by a digital recording system (Biopac Systems Inc, Santa Barbara, CA). Strips were stretched to 2 g (10 mN) and allowed to equilibrate for 30 minutes. Pyloric strips were then pretreated for 30 minutes with atropine (1 μmol/L; Sigma Chemical Co) and guanethidine (3.4 μmol/L; Sigma Chemical Co) to block cholinergic- and adrenergic-mediated responses, respectively. Non-adrenergic, non-cholinergic (NANC) relaxations were induced by electrical field stimulation (EFS; 90V, 2–16 Hz, 1-millisecond pulse for a duration of 1 minute). The NO dependence of the NANC relaxations was confirmed by incubation with the NOS inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 100 μmol/L; Sigma Chemical Co) for 30 minutes before EFS. To confirm the role of neuronal depolarization in evoking NANC relaxations, tetrodotoxin (TTX, 1 μmol/L; Calbiochem) was added to the bath. The weight of the tissue was measured at the end of each session.

Comparisons between the groups were performed by measuring the area under the curve of the EFS-induced relaxation (AUCR) for 1 minute and the baseline for 1 minute (AUCB) according to the following formula: (AUCR − AUCB)/Weight of Tissue (mg) = AUC/mg of Tissue.

Statistical Analysis 

Data are expressed as the mean ± SEM. Statistical analysis was performed with the aid of proprietary software (SigmaStat; SPSS Inc, Chicago, IL). Statistical significance was assumed if P < .05.

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Results 

Grafted NSCs Differentiate Into Neurons and Glia 

We have previously shown that NSCs can survive and differentiate when transplanted into the gastrointestinal tract.16, 17 Here we transplanted NSCs in the pyloric wall of nNOS−/− mice in combination with a caspase-1 inhibitor, Ac-YVAD-cmk, as previously described.17 One week after transplantation, GFP-positive NSCs were located in both submucosal and muscular layers and showed immunoreactivity for the markers PGP9.5 and GFAP, indicating the presence of both neuronal and glial phenotypes (Figure 1A and B). The number of neurons generated in the grafts outnumbered the glia by a ratio of approximately 4:1 (76% of GFP-positive cells also expressed PGP9.5, but only 20% expressed GFAP).

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  • Figure 1. 

    Transplanted NSCs survive in the pyloric wall of nNOS−/− mice and improve gastric emptying. (A) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week posttransplantation. GFP-positive NSCs are shown in green. PGP9.5 immunoreactivity is indicated in red. (B) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week posttransplantation. GFP-positive NSCs are shown in green. GFAP immunoreactivity is indicated in red. (A and B) lm, longitudinal muscle; cm, circular muscle. Scale bar = 20 μm. (C) Gastric emptying of liquids measured 20 minutes after gavage of dye in nNOS−/− mice 1 week following NSC transplantation or vehicle. *P < .01 by 2-tailed Student t test, 2-sample equal variance. Data are means ± SEM. n = 6 mice for vehicle and n = 8 mice for NSCs.

NSC Transplantation Improves Gastric Emptying to Liquids 

Adult transgenic mice lacking nNOS are characterized by a lack of pyloric relaxation leading to a grossly dilated stomach and severely delayed gastric emptying.4, 18 We measured gastric emptying of liquids 1 week following NSC transplantation in nNOS−/− mice. A control group of mice received intrapyloric injections of caspase-1 inhibitor only. Gastric emptying of a liquid feed was significantly accelerated in mice that received NSC transplantation as compared with vehicle controls (35% ± 3% in the vehicle group vs 50% ± 4% in the NSC group; P < .01 by Student t test) (Figure 1C). However, on gross examination, no overt change in gastric size or distention was noticeable.

NSC Transplantation Improves Relaxation of the Pyloric Sphincter 

EFS of ex vivo preparations of the pylorus under NANC conditions was performed to assess neurally mediated nitrinergic relaxation of the muscle. EFS-induced relaxation was significantly increased in pyloric muscle strips isolated from mice receiving NSCs as compared with control mice receiving vehicle alone (Figure 2A and B). This effect was completely blocked by the NOS inhibitor l-NAME (Figure 2C) and by TTX (Figure 3), confirming its nitrinergic nature and neural origin, respectively. Further, nNOS immunoreactivity was observed in the pylorus of animals within transplanted NSCs (Figure 4A and B) but not in control animals. The majority (73%) of GFP-positive NSCs expressed nNOS, a proportion very similar to that of those expressing the neuronal marker PGP9.5. VIP expression was not detected in grafted NSCs by immunohistochemical analysis (Figure 4C).

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  • Figure 2. 

    NSC transplantation results in increased relaxation of the pyloric muscle. (A) EFS of pyloric circular muscle under NANC conditions in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. The examples shown are from a representative experiment. (B) Quantification of NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. Analysis of the results by 2-way analysis of variance showed significant effects of both frequency (P < .001) and NSC transplantation (P = .012). Post hoc analysis with a Tukey multiple comparison test showed significant differences between the vehicle and NSC group at stimulation frequencies of 8 and 16 Hz. *P = .003; **P = .001. Data are means ± SEM. n = 7 mice for vehicle and n = 6 mice for NSCs. (C) Quantification of the effect of 100 μmol/L l-NAME on NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after vehicle injection or NSC transplantation. Data are means ± SEM. n = 7 mice for vehicle and n = 6 mice for NSCs.

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  • Figure 3. 

    Improved relaxation of the pyloric muscle is NO dependent and neurally mediated. Quantification of the effect of 1 μmol/L TTX on NANC-induced relaxations in response to EFS in nNOS−/− mice 1 week after NSC transplantation. Analysis of the results by 2-way analysis of variance for repeated measures showed a significant effect of TTX (P = .02). Post hoc analysis with a Tukey multiple comparison test showed significant differences in NANC-induced relaxations measured without (untreated) and in the presence of TTX at stimulation frequencies of 8 and 16 Hz. *P = .02; **P = .001. Data are means ± SEM. n = 7 for untreated and n = 6 for TTX.

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  • Figure 4. 

    Grafted NSCs express nNOS. (A) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. nNOS immunoreactivity is indicated in red. (B) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. Control obtained omitting the primary anti-nNOS antibody is shown in the middle panel. (C) Representative pictures of NSC grafts in cross sections of nNOS−/− mouse pylorus 1 week post-transplantation. GFP-positive NSCs are shown in green. VIP immunoreactivity would be in red, but it was absent in grafted cells. VIP immunoreactivity was found in intrinsic ganglia (data not shown). lm, longitudinal muscle; cm, circular muscle. Scale bar = 20 μm.

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Discussion 

We have previously shown that NSCs isolated from the fetal rodent brain can be transplanted in the gastrointestinal tract, where they differentiate into neurons and express nNOS.12 Here we report that NSC transplantation in the pyloric region of nNOS−/− mice significantly improves gastric emptying. This effect most probably results from the restoration of neurally mediated nitrinergic relaxation of the pyloric sphincter by nNOS-expressing transplanted neurons. Thus, relaxation of the pyloric sphincters from NSC-transplanted mice could be blocked by l-NAME. The fact that such relaxation could be induced by EFS strongly suggests that the NO is being produced by electrically excitable cells, the neuronal nature of which was further confirmed using TTX. Immunohistochemical staining with an antibody specific for nNOS further implicates this isoform as being responsible for the observed functional effects. By contrast, the other important inhibitory enteric neurotransmitter, VIP, is not expressed in grafted NSCs. Indeed, this is consistent with our data showing a complete blockade of EFS-induced relaxation by NOS inhibition alone. It is interesting to note that despite significant pyloric relaxation in vitro, gastric emptying was only partially improved, supporting a role for dysfunction of other regions of the stomach in the development of this phenotype, as has been suggested by others.4

Our results cannot completely exclude other concomitant effects that may also contribute to change in gastric function. We have previously shown that inflammatory infiltrates are recruited at the site of grafting of NSCs 1 week following transplantation,17 although these may become less prominent as time goes by.16 It is therefore possible that inflammatory mediators (including NO from inducible NOS) released locally could result in changes in smooth muscle function and hence gastric emptying. Moreover, it is possible that transplantation of NSCs may induce intrinsic inhibitory neurotransmitter activity in native tissue (eg, non-nitrinergic inhibitors such as VIPs or splice variants of nNOS itself that do not require the genetically deleted exon in this model). In this regard, a useful control experiment would have been the implantation of NSCs isolated from nNOS−/− mice, allowing us to address the specific contribution of the product of this gene on gastric emptying and pyloric function. Such an approach, however, requires a breeding colony for access to embryonic mice as well as hybridizing the nNOS−/− strain with a GFP-positive strain to facilitate post-transplantation tracking.

Central nervous system (CNS)-derived NSCs have been shown to generate neurons and glia in vitro and in vivo following implantation into the central and peripheral nervous system.11 When transplanted into the developing or adult CNS, they have been shown to undergo region-specific differentiation, generating mainly neurons in the hippocampus, dentate gyrus, and subventricular zone and mostly glia in the spinal cord.19, 20, 21 While the precise local factors responsible for these differences have not been identified, our results suggest that the gut environment provides a predominantly neurogenic drive for NSCs, with neurons outnumbering glia by a ratio of 4:1. Like their CNS counterparts, enteric glia serve as supportive elements for enteric neurons by creating a protective local microenvironment around the myenteric ganglia.22, 23 The significance of GFAP expression in transplanted grafts and the functional role of these cells need to be further investigated. However, we can speculate that they might play a supportive role for the neuronal grafts. In this respect, it is interesting to note that GFAP-expressing cells are located at the periphery of the grafted neuronal cells (Figure 1B), thus resembling the distribution of endogenous enteric glia in the myenteric plexus. Further studies are needed to address this issue.

Although in this report we used NSCs derived from the CNS, several other sources of NSCs may potentially be capable of achieving the same, if not better, results. These include NSCs derived from embryonic cells, neural crest stem cells, or their derivatives, enteric progenitor cells found in the immature gut. The advantages of using CNS-derived NSCs for transplantation in general include the fact that they have been the most extensively studied cell type for this purpose, they are easily isolated experimentally, and they are furthest along in terms of clinical trials.24 However, it is worth emphasizing that enteric neurons, although resembling neurons of the CNS in many ways, are derived from a distinct pool of precursor cells in early embryonic life and represent the end product of migratory stem cells originating in the neural crest. Recent data suggest that a small pool of neural crest–derived stem cells even persist in the adult gut.25 Using enteric neural precursors (ie, neural crest derivatives) therefore has the potential advantage of using cells of the same lineage as the desired phenotype based on the assumption that such cells are more “naturally poised” to generate a particular tissue26 and require little or no manipulation either in vitro or in vivo. However, the transplantation potential of enteric neural crest–derived stem cells has not been studied in vivo.

Finally, the developmental potentials and replicative capacity of pluripotent embryonic stem cells make them an attractive alternative for therapeutic cell replacement strategies. This benefit has to be balanced by the potential of differentiating a mixed population of differentiated cells and the risk of teratoma formation if planted directly. Recent studies do show, however, that neuronal precursors can be generated successfully from embryonic stem cells in vitro,27 and these in turn could be used for transplantation.

Although our findings are exciting, the short-term nature of our experiments must be acknowledged. We chose to study the effects of NSC transplantation at 1 week, because of the possibility of attrition of grafted neurons with the passage of time due to apoptosis or other processes, as we have recently shown.17 As in that study, we used a caspase inhibitor in these experiments as well, with the rationale to enhance NSC survival.

In summary, our data suggest that CNS-derived NSCs are capable of functionally replacing autonomic ganglia within the enteric nervous system. Although such techniques may work in short-term experiments, as represented by this study, many issues need to be addressed and techniques optimized before long-term cell replacement therapy in the gut can become a reality. These include not only establishing the optimal conditions for grafting and differentiation but also finding the best source of cells (CNS, neural crest, adult or embryonic). Nevertheless, this study clearly supports the potential of NSC transplantation as a valid therapeutic approach for disorders of the enteric nervous system.

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The authors thank Dr T. C. Savidge for critical review of the manuscript.

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 Supported by a grant from the National Institutes of Health (DK61423-01) and from the Moody Foundation.

PII: S0016-5085(05)01773-7

doi:10.1053/j.gastro.2005.08.055

Gastroenterology
Volume 129, Issue 6 , Pages 1817-1824, December 2005

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