Neural stem cells express RET, produce nitric oxide, and survive transplantation in the gastrointestinal tract

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RAPID COMMUNICATIONS Neural Stem Cells Express RET, Produce Nitric Oxide, and Survive Transplantation in the Gastrointestinal Tract MARIA–ADELAIDE MICCI,* RANDALL D. LEARISH,‡ HUI LI,* BINCY P. ABRAHAM,* and PANKAJ JAY PASRICHA* *Enteric Neuromuscular Disorders and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston, Texas; and the ‡Promega Corporation, Madison, Wisconsin

Background & Aims: Transplantation of neural stem cells (NSC) has been shown to be successful in a variety of experimental models of nongastrointestinal diseases. The aim of this study was to assess the potential of NSC transplantation as a therapeutic strategy for neuronal replacement in disorders of the enteric nervous system. Methods: Central nervous system-derived NSC (CNSNSC) were obtained from the subventricular zone of rat brain (E17). Expression of RET, GFR␣1, and neuronal nitric oxide synthase (nNOS) was assessed by Western blot and immunocytochemistry. Nitric oxide (NO) production was assessed using the NO-sensitive fluorescent indicator DAF-2. CNS-NSC (labeled with CM-DiI) were transplanted into the pylorus of mice and fluorescent double-labeling immunostaining for ␤III-tubulin or PGP 9.5 and nNOS was performed at 2, 4, and 8 weeks after transplantation. Results: Our results show that CNS-NSC express both the receptors (RET and GFR␣1) for the enteric neurotrophin, GDNF; GDNF, in turn, induces expansion of the RET-expressing CNS-NSC population. Furthermore, CNS-NSC express nNOS and produce NO in vitro. When transplanted into the gut, CNS-NSC differentiate into neurons, continue to express nNOS and survive at least 8 weeks. Conclusions: We conclude that transplantation of CNS-NSC bears promise as a potential cellular replacement strategy for enteric neurons.

eural stem cells (NSC) are primordial uncommitted cells that give rise to an array of specialized cells in the central nervous system (CNS). NSC have been identified in various anatomical sites in the brain including the subventricular zone (SVZ).1– 6 Following implantation in vivo, NSC have been shown to develop into cells of the neuronal and glial lineage and to have the potential for treating a wide variety of focal, as well as diffuse, neurodegenerative conditions.7–9 Recent studies have suggested that NSC are also capable of differentiating into hematopoietic and skeletal muscle lines.10,11 Furthermore, it has been shown that NSC from the adult

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mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers, populating even liver, stomach, and intestines.12–13 Thus, NSC have a very broad developmental capacity and may be used to generate a multitude of cell types for transplantation in a variety of diseases. These properties led us to hypothesize that these cells may also be successful in restoring neuronal populations within the enteric nervous system (ENS) in conditions associated with loss of neurons in the myenteric plexus such as congenital hypertrophic pyloric stenosis, Hirschsprung’s disease, and achalasia.14 –15 Although little is known about the factors that maintain a healthy ENS in adult animals, it is becoming clear that normal development of the ENS is determined principally by interaction of the receptor tyrosine kinase (RET) with the neurotrophic growth factor, GDNF (glial-derived neurotrophic factor).16 This is mediated by extracellular glycosylphospatidylinositol-linked molecules: the GDNF family receptor (GFR) ␣1 and ␣2, that confer specificity for GDNF and neurturin respectively.17 The aim of this study was to characterize the therapeutic potential of CNS-derived NSC (CNS-NSC) as candidate transplant cells for the ENS. We pursued this by assessing the ability of CNS-NSC to produce nitric oxide (NO), to respond to the enteric neurotrophin GDNF, and to survive in the gastrointestinal tract after transplantation. Our data shows that CNS-NSC express neuronal nitric oxide synthase (nNOS) and produce NO in vitro. FurAbbreviations used in this paper: bFGF, fibroblast growth factor; EGF, epidermal growth factor; ENS, enteric nervous system; GDNF, glial-derived neurotrophic factor; GFR␣1, GDNF family receptor ␣1; L-NAME, N-nitro-L-arginine methyl ester; nNOS, neuronal nitric oxide synthase; NSC, neural stem cells; PFA, paraformaldehyde; RET, receptor tyrosine kinase; SDS, sodium dodecyl sulfate; SVZ, subventricular zone; TBST, Tris-buffered saline containing Tween. © 2001 by the American Gastroenterological Association 0016-5085/01/$35.00 doi:10.1053/gast.2001.28633

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ther, CNS-NSC express a functional receptor system for the enteric neurotrophin GDNF (RET and GFR␣1) in vitro. Finally, we report that CNS-NSC can be successfully transplanted into the mouse gastrointestinal tract, where they appear to survive for several weeks, differentiate into neurons and express nNOS.

Materials and Methods Animals Staged-pregnant female Holtzman rats (Harlan Sprague Dawley Inc., Cumberland, IN) at embryonic day 17 were used for the isolation of CNS-NSC. Adult male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) 20 gm, were used in all transplantation experiments. Experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch, Galveston, in accordance with the guidelines provided by the National Institutes of Health.

Generation and In Vitro Culture of Rat CNS-NSC Cell culture reagents were obtained from Gibco-BRL (Gaithersburg, MD) except where noted. Staged-pregnant female Holtzman rats at embryonic day 17 were deeply anesthetized with sodium pentobarbital (70 mg/kg, intraperitoneally [IP]) and a midline incision was made to expose the embryos. The brains of embryonic rats were removed and the SVZ tissue was dissected from each brain hemisphere, as previously described.18 Single cell suspensions were then made from this tissue using dispase/trypsin treatment and gentle trituration. The fractions were combined, pelleted, and resuspended in Neurobasal medium containing B27, 2 mmol/L glutamine, and penicillin-streptomycin (NB27). After 2 to 4 hours, the cells were centrifuged and media replaced with NB27 plus 20 ng/mL fibroblast growth factor (bFGF) and 20 ng/mL epidermal growth factor (EGF) (Promega, Madison, WI). Under these conditions, embryonic CNS-NSC can be propagated in culture for several weeks retaining their undifferentiated state.18

Western Blotting Except where indicated all reagents were obtained from Sigma (St. Louis, MO). Total protein extract was obtained from CNS-NSC by lysing the cells in buffer A consisting of 2% sodium dodecyl sulfate (SDS), 100 ␮mol/L protease cocktail inhibitor, 1 mmol/L phenylmethylsulphonyl fluoride, 1 mmol/L EDTA in 50 mmol/L Tris-buffered saline (TBS, 50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl). Insoluble material was removed by centrifugation at 13,000 ⫻g for 10 minutes. Rat brain was homogenized in Buffer A and centrifuged at 13,000 g for 10 minutes. nNOS. Total proteins were diluted in 4⫻ SDS loading buffer (3g Tris; 8g SDS; 2.5g dithiothreitol (DTT); 0.05g Bromophenol blue; 40% (by volume) glycerol per 100 mL

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buffer), boiled 5 minutes, and loaded into a 4%–15% TrisHCl gel. After electrophoresis (1.5 hours at 40 mA), the proteins were electrotransferred onto nitrocellulose membrane overnight. After preblocking for 1 hour in 5% milk in TBS with 0.1% Tween 20 the membranes were incubated with 1:1000 dilution of rabbit polyclonal anti nNOS antibody (Transduction Laboratories, Lexington, KY) followed by incubation with 1:5000 dilution of horseradish peroxidase (HRP)conjugated goat anti rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA). RET. Five hundred micrograms of protein were incubated with 2 ␮L of anti-RET antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 ␮L of protein A-agarose (Boehringer Mannheim, Indianapolis, IN) at 4°C overnight. Precipitated aggregates were resuspended in Laemmli’s sample buffer and run on a 7.5% SDS-PAGE gel, transferred to PVDF membranes (Amersham, Buckinghamshire, England) and incubated with anti-RET antibody at a dilution of 1:1000 in 5% milk in TBS with 0.05% Tween 20 for 2 hours at room temperature, followed by incubation with an HRP-conjugated secondary antibody. As a negative control, the samples were treated in the same manner as described previously with the omission of the primary antibody in immunoprecipitation. GFR␣1. For the detection of GFR␣1, total protein extract was obtained from CNS-NSC by lysing the cells in buffer composed of 50 mmol/L 3-(N-morpholino) propanesulfonic acid buffer pH 8.6, 1% Nonidet-40 detergent (Fluka Biochemica, Milwaukee, WI), 250 mmol/L sodium chloride, 1 mmol/L dithiothreitol (DTT), 10 ␮g/mL aprotinin, 10 ␮g/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, and 3 mmol/L ethylene glycol-bis (␤-aminoethyl-ether)-N,N,N⬘,N⬘tetraacetic acid. Loading buffer was added to 10 ␮g of lysate, and the sample was electrophoresed through 4%–20% precast polyacrylamide gels (Novex, San Diego, CA). The proteins were transferred to nitrocellulose blots, blocked with Tris pH 7.8-buffered saline containing 0.05% Tween (TBST), and 1% bovine serum albumin (BSA), and then probed with mouse monoclonal antibody to GFR␣1 (1:5000, Transduction Laboratories, Lexington, KY) in a solution containing TBST and 0.5% BSA for 1.5 hours. Then, the blot was washed 3 times with TBST and incubated in HRP-conjugated IgG secondary antibody (Promega, Madison, WI) at a dilution of 1:20,000 for 1 hour. Immunoreactive bands were detected by a chemiluminescent Western blot detection kit (Amersham, Buckinghamshire, England), according to the manufacturer’s instructions. Results were analyzed by a computer-assisted image analysis system (Alpha Innotech, San Leandro, CA).

Immunocytochemistry CNS-NSC were seeded onto poly-ornithine-coated chambered slides (Nunc, Naperville, IL). Cells were fixed with 100% methanol for 10 minutes at ⫺20°C. CNS-NSC were blocked with 5% normal serum for 1 hour at room temperature, washed in phospate-buffered saline (PBS), and incubated with primary antibodies diluted in PBS containing 1.5%

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Figure 1. CNS-NSC express nNOS and produce NO in vitro. (A ) Western blot analysis of total rat pituitary lysate (lane 1, as positive control) and rat CNS-NSC total proteins extract probed with (lane 2) or without (lane 3, as negative control) a specific anti-nNOS antibody. (B) nNOS immunoreactivity in rat CNS-NSC in culture for 24 hours. (C ) NO production in CNS-NSC. The relative fluorescence intensity reflecting NO production by CNS NSC was measured in the presence of L-arginine (1 mmol/L; n ⫽ 11 cells) or L-NAME (100 ␮mol/L; n ⫽ 8 cells) at 1, 2, 3, and 4 minutes after addition of ionomycin (1.4 ␮mol/L). A significant increase in fluorescence intensity is observed in the presence of L-arginine as compared with L-NAME. Data are from one experiment but are representative of 3 others. Error bars indicate SD. * P ⬍ 0.05; ** P ⬍ 0.01.

normal serum overnight at 4°C. The following antibodies were used: anti-PGP 9.5 (1:500 dilution; Neuromics Inc., Minneapolis, MN), anti-␤III-tubulin (0.5 ␮g/mL; Promega, Madison, WI), anti-GFAP (1:400 dilution; Chemicon International Inc., Temecula, CA), anti-nNOS (1:400 dilution; Transduction Laboratories, Lexington, KY), anti-GFR␣1 (1:400 dilu-

tion; Transduction Laboratories, Lexington, KY), and antiRET (kindly provided by Dr. David Anderson, California Institute of Technology, Pasadena, CA). Cells were then incubated with the appropriate Alexa-conjugated secondary antibody (Alexa-488; Alexa-594: 1:500 dilution; Molecular Probes, Eugene, OR) for 1 hour at room temperature. Cell

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Figure 2. Expression of nNOS in differentiated rat CNS-NSC in vitro. (A–C) Photomicrographs of cultured rat CNS-NSC double-stained for (A ) ␤III-tubulin, (B) nNOS, and (C ) superimposition of both. (D–E) Photomicrographs of cultured rat CNS-NSC double-stained for (A ) PGP 9.5, (B) nNOS, and (C ) super-imposed of both. Note that nNOS immunoreactivity is present in all the ␤III-tubulin and PGP 9.5-positive cells, indicating that nNOS expression is maintained in differentiated neurons. (G–I) Photomicrographs of cultured rat CNS-NSC doublestained for (A ) GFAP, (B) nNOS, and (C ) superimposition of both. nNOS immunoreactivity is absent in GFAP-positive cells (arrows). Calibration bars: 20 ␮m.

nuclei were counterstained with Vectashield mounting medium containing 4⬘,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Controls were produced by omitting the primary antibodies.

Cell Counts and Image Analysis Immunolabeled CNS-NSC were visualized and images captured using an Olympus BX60 microscope (Olympus,

Figure 4 (continued on next page).

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Melville, NY) equipped with fluorescence and digital imaging with a cooled CCD camera (Photometrics CoolSNAP, Roper Scientific) and Metaview software (Universal Imaging Corp., West Chester, PA). A total cell count per field was achieved automatically by applying software filters that identified DAPI-stained nuclei based on 2-dimensional area and pixel intensity, and adjustments were made for overlapping nuclei. Immunolabeled cells were counted by manual tagging.

Nitric Oxide Detection CNS-NSC were plated onto poly-ornithine-coated 25 mm glass coverslips. The cells were incubated for 1 hour at 37°C in standard Krebs solution (130 mmol/L NaCl, 5.5 mmol/L KCl, 2.5 mmol/L CaCl2 , 1.25 mmol/L MgCl2 , 10 mmol/L glucose, 13 mmol/L sucrose, 20 mmol/L HEPES [pH 7.3]) containing the membrane-permeant NO-sensitive fluorescent indicator DAF-2/DA (10 ␮mol/L, Alexis Biochemicals, San Diego, CA). After loading, the cells were washed, placed in Krebs solution, and viewed with a confocal laser-scanning microscope system (Noran Instrument, Madison, WI), using an argon-ion laser (488 nm) coupled to an inverted Nikon Diaphot microscope (Nikon Inc., Dallas, TX). The medium was then switched to Krebs solution containing L-arginine (1 mmol/L; Sigma, St. Louis, MO) or the nNOS inhibitor N-nitro-L-arginine methyl ester (L-NAME: 100 ␮mol/L; Sigma, St. Louis, MO). Cells were incubated for 10 minutes and then the calcium ionophore ionomycin (1.4 ␮mol/L; Calbiochem-Novabiochem Corp., La Jolla, CA) was added to the cells to activate nNOS.

Labeling Methods and Preparation of Cells for Transplantation To enable the detection of the cells in vivo, CNS-NSC were labeled with CM-DiI (Molecular Probes, Eugene, OR) according to manufacturer’s instructions. After washing in phosphate-buffered saline (PBS), the cells were resuspended in PBS at a concentration of 400,000 cells/␮L and kept on ice.

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Male adult mice were deeply anesthetized with sodium pentobarbital (70 mg/kg, intraperitoneally [IP]). A midabdominal incision was made and the pylorus was identified. Two microliters of CNS-NSC suspension were injected bilaterally into the pyloric wall using a 22-gauge needle attached to a 10 ␮L Hamilton syringe.

Tissue Processing At 2, 4, and 8 weeks after CNS-NSC transplantation, mice were deeply anesthetized with sodium pentobarbital (70 mg/kg, IP), transcardially perfused and fixed with ice-cold 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4). The pylorus was removed, postfixed in 4% PFA for 1 hour at room temperature, and cryoprotected by infiltration in 30% sucrose solution in PBS overnight at 4°C. The tissue was placed in OCT embedding medium (Tissue Tek, Sakura, Tokyo) and rapidly frozen over dry ice. Frozen sections (15 ␮m thick) were cut at –15°C on a cryostat (TBS, Durham, NC), placed on gelatin-coated slides (VWR, West Chester, PA) and stored at – 80°C until needed. For double-immunofluorescence staining, sections were permeabilized with 50% glycerol/50% PBS, blocked with 5% normal serum for 1 hour at room temperature, washed in PBS, and incubated with primary antibodies diluted in PBS containing 1.5% normal serum, overnight at 4°C. The following antibodies were used: anti-PGP 9.5 (1:500 dilution), anti-␤III-tubulin (0.5 ␮g/mL), anti-GFAP (1:400 dilution), and anti-nNOS (1:400 dilution). After washing, sections were incubated for 1 hour at room temperature with Alexa-conjugated secondary antibodies (Alexa-488 and Alexa-350: 1:500 dilution; Molecular Probes, Eugene, OR).

Statistical Analysis Data were expressed as the mean ⫾ standard error (SE) or standard deviation (SD). Two-tailed Student t test was used for the comparison between 2 independent groups.

Figure 4. RET immunoreactivity in rat CNS-NSC that were treated for 10 days with either (A ) NB27 media plus 20 ng/mL EGF and 20 ng/mL bFGF or (B) the same media plus 100 ng/mL GDNF. Cell nuclei are counterstained in blue with DAPI. Calibration bars: 20 ␮m. (C ) The histogram demonstrates the significant increase in the percentage of RET-positive cells under the effects of 100 ng/mL GDNF as compared with control media (control). Error bars represent SE. *P ⫽ 0.002. Total number of cells: n ⫽ 2350 (control); n ⫽ 2417 (GDNF) pooled from 3 independent experiments.

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Results CNS-NSC Express nNOS and Produce NO In Vitro Immunoblot analysis of proteins extracted from CNS-NSC and immunocytochemistry showed that nNOS is expressed in these cells (Figure 1A and B). Bioimaging of cultured CNS-NSC loaded with a NO-sensitive fluorescent indicator showed a time-dependent increase in fluorescence intensity in the presence of the nNOS substrate L-arginine (1 mmol/L), an effect that was suppressed by the nNOS inhibitor L-NAME (100 ␮mol/L) (Figure 1C). Previous studies have shown that upon withdrawal of the mitogens EGF and FGF, CNS-NSC differentiate into neurons and astrocytes.18 To assess whether nNOS expression is maintained in differentiated neurons, CNSNSC were cultured for 7 days in the absence of the mitogens EGF and FGF. Double immunofluorescence staining for nNOS combined with either GFAP (a marker for astrocytes) or 1 of 2 widely used neuronal markers, ␤III-tubulin and PGP 9.5 (a more specific marker for mature neurons) revealed that expression of this enzyme is maintained in differentiated neuronal cells (Figure 2, A–F ) but not in glial cells (Figure 2, G–I ). CNS-NSC Express RET and GFRa1 and Respond to GDNF In Vitro To test whether CNS-NSC would be responsive to local growth factors, we examined the expression of the receptor system for the enteric neurotrophin GDNF. Cultured CNS-NSC express both of the GDNF coreceptors, RET and GFR␣1 (Figure 3). When CNS-NSC were cultured in the presence of GDNF (100 ng/mL) for 10 days, the number of RETpositive cells increased significantly as compared with control (60% vs. 22.8%; P ⫽ 0.002) (Figure 4). CNS-NSC Can Be Successfully Transplanted Into the Pyloric Wall DiI-labeled CNS-NSC were injected into the pyloric wall of adult mice and the tissue was analyzed at 2, 4, and 8 weeks as described in Methods. DiI-labeled CNS-NSC were recovered in the host tissue at each time point examined. Cells appeared not to have migrated from the site of injections and were situated between the longitudinal and circular muscle layer in close proximity to the myenteric plexus in the host tissue (Figure 5A). To assess whether grafted CNS-NSC differentiate into neurons and express nNOS, we performed double immunofluorescence staining for the neuronal markers ␤IIItubulin or PGP 9.5 and for nNOS. Our results show that

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nNOS expression is maintained in a subpopulation of grafted cells that is also positive for the neuronal marker PGP 9.5, as well as ␤III-tubulin (Figure 5B and C).

Discussion Gastrointestinal motility disorders such as achalasia, congenital hypertrophic pyloric stenosis, and Hirschsprung’s disease are characterized by complete or partial loss of NO-producing neurons in the ENS, a well-defined system of neurons that regulates several aspects of gastrointestinal physiology including motility and secretion.14,15,19 The loss of nitrinergic neurons leads to the inability of the gastrointestinal smooth muscle to relax.20 –21 In achalasia, treatment consists of compensating for the loss of the inhibitory neurons by procedures that weaken the muscle such as myotomy or dilation,15 whereas surgical intervention is frequently the only option in congenital hypertrophic pyloric stenosis and Hirschsprung’s disease. Although partially successful, these methods do not really address the underlying problem. Theoretically, the only hope for a real cure for some of these conditions is replacement of the lost population of neurons. Until now this was an elusive goal, but recent advances in the field of NSC transplantation, particularly in the CNS, have rendered it more feasible.7,22–24 NSC are characterized by a broad developmental capacity and may be used to generate a variety of cell types, including neurons.6,25 NSC have been isolated from the embryonic, neonatal, and adult mammalian nervous system and propagated in vitro by a variety of means.11,26 Recent studies have shown that other stem cells, such as adult rat and human bone marrow stromal cells25 and neural crest stem cells27 can also be induced to differentiate into neurons. A remarkable feature of NSC is their ability to respond in a differential manner, depending on the nature of local cues, to produce phenotypes that are characteristic and appropriate for the environment they are transplanted into, without disrupting the normal dynamics of the target tissue.23,24,27,28 This lead us to hypothesize that NSC may be used to restore nitrinergic neuronal function when transplanted into the ENS. A critical requirement for NSC transplantation to succeed clinically is the ability of the transplanted cells to express functionally significant amounts of the deficient neurotransmitter (i.e., NO). Previous studies have reported the expression of nNOS in neural progenitor cells of murine and human origin.29 –30 Our first goal was therefore to assess the capability of CNS-NSC to express nNOS. Our data show that nNOS is expressed in rat CNS-NSC and that this enzyme is functional in these cells in vitro. Most importantly, our data show that nNOS expression is

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Figure 3. CNS-NSC express the receptor complex for GDNF. (A ) Western blot analysis of total proteins extracted from rat brain (lane 1, as positive control) and rat CNS-NSC probed with (lane 2) or without (lane 3, as negative control) a specific anti-RET antibody. (B) RET immunoreactivity in cultured rat CNS-NSC. Specific immunoreactivity is observed in a subpopulation of cells. (C ) Western blot analysis of total proteins extracted from rat brain (lane 1, as positive control) and rat CNS-NSC with (lane 2) or without (lane 3, as negative control) anti-GFR␣1 antibody. (D) GFR␣1 immunoreactivity in cultured rat CNS-NSC. Panels B and D show superimposition of fluorescent and bright field images.

maintained in these cells during neuronal differentiation both in vitro and in vivo. Having established the expression of functional nNOS, we further explored the potential of CNS-NSC as cell replacement candidates in the ENS, by assessing their ability to respond to putative gut-derived neurotrophic factors. In this context, we focused on GDNF that, along with neurturin, appears to be the principal determinant by which the microenvironment of the gut influences the proliferation and

differentiation of enteric neuronal precursors. 31 Here we have shown that CNS-NSC not only express the receptor complex for GDNF (RET and GFR␣1), but also respond to exogenous GDNF with an expansion of the RET-positive population. Thus, these cells clearly possess the ability to respond to at least one of the putative gut-specific environmental signals, making them excellent candidates for future studies on stem cell transplantation in the gastrointestinal tract.

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Although the concept of using enteric neuronal precursors to repopulate the ENS has been previously demonstrated in vitro,32 no information on their use in vivo has been so far available. We have now shown that CNS-NSC can survive in vivo in the host mouse gastrointestinal tissue up to 8 weeks postgrafting, and that at least a subpopulation of implanted cells differentiates into neurons and express nNOS. In addition to differentiated neurons, a few scattered glial cells (identified by GFAP staining) were also observed, but these did not express nNOS (results not shown). This is in keeping with our in vitro data showing that nNOS expression is maintained in CNS-NSC after neuronal differentiation but not in glial cells. However, further studies are needed to more precisely characterize the identity and fate of transplanted rat CNS-NSC, including both neuronal and nonneuronal cell types. Transplanted CNS-NSC were observed in close proximity to the injection site at all the time point studied, indicating that little or no migration of the grafted cells occurs in the host tissue. No evidence of significant inflammation was noticed, despite the xenogeneic nature of the transplants. This is in keeping with other reports that attest to the low immunogenic potential of such cells.23 However, clearly this issue needs to be addressed in further studies that more precisely quantify the survival and migration of transplanted CNS-NSC and other posttransplantational events over a longer period of time. In conclusion, our results show for the first time that NO-producing CNS-NSC survive within the pyloric wall after transplantation, possibly by responding to the enteric neurotrophin GDNF. Taken together, these data support the hypothesis that transplantation approaches may be of potential therapeutic benefit in pathological gastrointestinal conditions associated with the loss of NO-producing neurons.

Š Figure 5. (A ) DiI-labeled CNS-NSC 8 weeks after transplantation into the pyloric wall of C57BL/6J mice. Cross sections of mouse pylorus were counterstained in blue with DAPI to identify cell nuclei. To help identify the location of the grafted cells, bright field images (not shown) were superimposed with fluorescent images. DiI-labeled cells are recovered in the mouse pyloric wall where they are mainly localized between the longitudinal (LM) and circular (CM) muscle layer. (B-C ) Double immunofluorescence staining of grafted DiI-labeled CNSNSC shows colocalization of DiI with nNOS and the neuronal markers ␤III-tubulin (B) and PGP 9.5 (C ), indicating that at least a subpopulation of grafted cells (arrows) differentiate into neurons and maintain the expression of the enzyme nNOS. Calibration bars: 10␮m. LM, longitudinal muscle; CM, circular muscle.

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Received June 13, 2001. Accepted August 7, 2001. Address requests for reprints to: Pankaj Jay Pasricha, M. D., Enteric Neuromuscular Disorders and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0764. e-mail: [email protected]; fax: (409) 772-4789. Supported by a grant from the Texas State Agency as part of the Advanced Research Program. The authors thank the laboratory of Dr. Giulio Taglialatela for assistance in performing the Western blotting for nNOS, Dr. Hiroki Toma for assistance in performing the Western blotting for RET, Dr. David Anderson (California Institute of Technology, Pasadena, California) for providing anti-RET antibody, Brenda Kenworthy for skillful technical assistance and Dr. W. A. Hoogerwerf for helpful discussion and critical review of this manuscript. The University of Texas Medical Branch has applied for a patent on the use of stem cells for gastrointestinal disorders.