Gastroenterology

Gastroenterology

Volume 121, Issue 4, October 2001, Pages 757-766
Gastroenterology

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

https://doi.org/10.1053/gast.2001.28633Get rights and content

Abstract

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 (CNS-NSC) were obtained from the subventricular zone of rat brain (E17). Expression of RET, GFRα1, 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 βIII-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 GFRα1) 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.

GASTROENTEROLOGY 2001;121:757-766

Section snippets

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

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).

. 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

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

Acknowledgements

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.

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    ☆

    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 University of Texas Medical Branch has applied for a patent on the use of stem cells for gastrointestinal disorders.

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    Copyright © 2001 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.