| Recently more and more attention has been paid to the studies associated with stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types. According to their capacity of differentiation, stem cells have been divided into three major groups: totipotential stem cells, multipotential stem cells and unipotential stem cells. It was believed that only embryonic stem cells derived from the inner cell mass could differentiate into any cell type and belong to totipotential stem cells, but adult stem cells or tissue-specific stem cells could only differentiate into cells of the tissue of origin and belong to multipotential stem cells or unipotential stem cells. This finding is important for the development of cytobiology theory and cell transplantation treatment for many diseases.A neural stem cell (NSC) can be most simply defined as any cell which is capable of self-renewal for extended periods of time, the progeny from which is capable of forming the components of nervous tissue, differentiating into neurons, astrocytes and oligodendrocytes. NSCs have been isolated from several sites from fetal, neonatal or adult mammalian animal and human central nervous system (CNS), such as sub ventricular zone (SVZ), dentate gyrus of hippocampus, olfactory bulb and granular layer of cerebellum. With the recognition that NSC, propagated in culture, could be reimplanted into mammalian brain where they could reintegrate appropriately, stem cell therapy is the new strategy to treat a variety of diseases, through replacing, repairing the injured tissue or organ, then strengthening the function of them. The early-formed neural tube consists of proliferating, morphologically homogeneous cells, termed "neuroepithelial stem cells (NESCs)" which generate neurons, astrocytes, and oligodendrocytes through a series of intermediate precursor cells. NESCs can develope into CNS and peripheral nervous system (PNS) derivatives, which illustrates that NESCs are the most primitive neural stem cells. The advantages of using embryonic NESCs for transplantation in general include the fact that they have been an extensively studied cell type for this purpose, they are easily isolated experimentally, and in previous in vivo transplant studies have demonstrated their good survival and vigorous neuronal differentiation tendency in the host environment, unlike other sources of NSCs.Aganglionic megacolon, i.e. Hirschsprung's disease (HD), is a functional intestinal obstruction with an incidence of 1/3000～1/5000 live births. This disorder is characterized by severe constipation due to the absence of enteric ganglia along a variable length of the intestine. Varying lengths of the distal colon are unable to relax, causing functional colonic obstruction over time. Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children. With regard to the treatment of aganglionic gut conditions, surgical intervention of removal of the affected segments is frequently the only option, whereas surgical therapy does not always lead to a complete recovery and restoration of all bowel functions, which followed by some complications such as constipation, fecal incontinence and most serious enterocolitis, colonic rupture and psychic problem.The enteric nervous system (ENS) is a well-defined system of neurons and glial cells that regulates several aspects of gastrointestinal physiology, including motility and secretion. The ENS descends from migrating neural crest cells and is structured into different plexuses embedded in the gastrointestinal tract wall, including myenteric plexus and submucosal plexus. The neural crest, which forms at the dorsal tips of the neural epithelium, gives rise to migratory cells that colonize a wide range of embryonic tissues. Neural crest cells, at the human embryonic 5-12 weeks, migrating on precisely controlled pathways from the neural tube into the developing gut, function as enteric nervous system stem cells and give rise to all neuronal and glial subtypes found in the gastrointestinal tract. Disturbances appearing during this time affect the proper formation and/or the proper functioning of the ENS, thus leading to severe intestinal dysfunctions, e.g., Hirschsprung's disease.The lack of effective therapies for these syndromes due to loss of ENS has led us to explore the possibilities of novel approaches such as cell transplantation. Using benzalkonium chloride (BAC) ablation of the ENS-induced aganglionic megacolon animal model, we addressed this issue in NESCs transplantation research for enteric nervous system repair, followed the morphological fate of the grafted cells and evaluation of the enteric motility recovery. This study can be divided into three parts. Part one: Isolation, culture and labelling of neuroepithelial stem cells from neural tube in vitroIsolation, culture and labelling of NESCs are the premise of cell transplantation study. The NESCs were isolated from neural tubes of E11.5d Wistar rats. By microdissection and mechanical trituration, a cell suspension was obtained and resuspended in neurobasal medium containing B27, plus bFGF. By 72 hours under non-adherent culture conditions, dozens of NESCs had proliferated to form neurosphere-like aggregates and a neurosphere consisted of hundreds of cells developed after cultured for 6 days. The generated neurospheres were characterized as Nestin positive. In the presence of serum, migrating cells from neurosphere differentiated into microtubule associated protein 2 (MAP2)-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. The mRNA from cultured NESCs expressed the receptor system for the enteric neurotrophin- rearranged during transformation (RET) receptor tyrosine kinase and GDNF family receptorα1 (GFRα1) through RT-PCR, indicating that these cells would be responsive to enteric local growth factors and be more suitable to be candidate transplant cells for the enteric nervous system. To label grafted cells, cultures were incubated with 5-bromo-2-deoxyuridine (BrdU) for 72 hours. After incorporation with BrdU, approximately 90% cells in a neurosphere could be labelled with BrdU, which could also be detected after differentiation of the neurosphere. BrdU labelling will provide astable method for tracing grafted cells in vivo.Part two: Establishment of the rat model for experimental aganglionicmegacolonAccording to the typical pathophysiology of aganglionic megacolon, we applied 0.5% BAC to the serosal surface of the rat colon for 30min to ablate the ENS selectively. Four weeks after BAC treatment, autopsy revealed a narrow segment at the site of BAC treatment and marked dilation of the proximal segment. Inflation stimulated contraction was abolished in the BAC treatment group. By histologic examination, the portion treated with BAC was confirmed to be denervated and lack of acetylcholinesterase activity; whereas there were normal myenteric and submucous plexuses and acetylcholinesterase activity in the controls. The expression of neuronal nitric oxide synthase (nNOS) and choline acetyltransferase (ChAT) decreased dramatically after BAC treatment through Western blot analysis. Aganglionic megacolon was produced successfully in the rat by applying BAC to ablate enteric plexus. This model will provide the basis for further studies on etiology, pathophysiology and new treatment of aganglionic megacolon.Part three: The assessment of neuroepithelial stem cells after transplantation into the aganglionic colon of ratOn the basis of successful culture NESCs in vitro and establishment an aganglionic megacolon model, we elucidated the possibility of intracolonic grafting of NESCs as a therapeutic strategy for enteric neuronal replacement. BrdU-labelled NESCs were transplanted into the denervated colon. After transplantation, differentiation of grafted cells was examined and the intestinal motility was assessed. Our results indicate that when transplanted into the denervated gut, NESCs survived and could differentiate into protein gene product 9.5 (PGP9.5)-positive neurons and GFAP-positive glial cells in vivo at 4-week assessment. Further assessment for neurochemical markers at the 8-week stage showed some donor-derived cells were positive for nNOS and ChAT. In addition, inflation stimulated contraction and electrical field stimulation (EFS)-induced response were observed in NESCs grafted group compared with no reaction in denervated group. NESCs can survive,differentiate into neuronal phenotypes and improve denervated colon motilityrecovery in vivo, which indicates that NESCs provide a promising cellular replacement candidate for ENS.ConclusionsIn summary, we successfully set up an aganglionic megacolon rat model, cultured, labelled NESCs and transplanted NESCs into denervated colon. Furthermore, our results showed that NESCs could survive and function in the aganglionic colon wall after transplantation. The method described here may be a valuable tool in creating great therapeutic value of NESCs that can be used in disorders of enteric nervous system such as aganglionic megacolon.
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