| Stem cells are specialized cell types in the embryonic and adult tissue.They have the ability of long-term self-renewal and can differentiate into one or more specialized cell types in specific condition.According to their capacity of differentiation,stem cells have been divided into three major groups:totipotent stem cells,multipotent stem cells and unipotent stem cells.The fertilized mammalian egg is totipotent because it can give rise to the entire organism.The inner cell mass of the blastocyst which consists of pluripotent stem cells can give rise to virtually every type of cell except the trophoblast in the body and infinitely proliferate in vitro.These pluripotent stem cells give rise to more specialized cells that are committed to specific lineages such as blood,nervous system,etc.,and are called multipotent stem cells. They are present in many tissues of fetal and adult animals and are important in tissue repair and homeostasis.Good examples include hematopoeitic stem cells(HSC) and neural stem cells(NSC).The ability of multiple lineage differentiation and long term self-renewal make stem cells to be the most important "seed cells" in future regenerative study.Stem cells have come to occupy a permanent important place on the front page for their therapeutic impact.Neural stem cells(NSCs) can be defined as cells which display the ability to self-renew and give rise predominantly to neurons and glia(astrocytes and oligodendrocytes).NSCs have been identified and isolated from various sites of fetal, neonatal or adult mammalian animal and human central nervous system(CNS) and peripheral nervous system(PNS),including the cerebellum,cerebral cortex, hippocampus,olfactory bulb,subventricular zone and spinal cord.Since cortex-originated neural stem cells have a very broad developmental capacity and may generate a multitude of cell types,a great deal of interest has focused on the potential therapeutic applications of them and they have emerged as a possible donor material aimed at neural transplantation for the repair of damaged neural circuitry. The advantages of using embryonic cortex-originated neural stem cells for transplantation in general include that they have been an extensively studied cell type for this purpose,and they are easily isolated experimentally.Many results of previous in vivo transplant studies have demonstrated their good survival and vigorous neuronal differentiation tendency in the host environment.The enteric nervous system(ENS) is a relatively autonomous collection of neurons and supportive cells that is present in the gut wall and regulates the activity of the gastrointestinal tract,including secretion,absorption,and motility.The neural elements of the ENS come into being ganglionated plexuses located within the gut wall in two relatively distinct collections found either submucosally(submucosal plexus) or between the muscle layer(myenteric plexus).The ENS descends from the neural crest.Neural crest cells,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 and disorders occuring during this time and the process would affect the proper formation and/or the normal function of the ENS,and consequently lead to various severe intestinal dysfunctions,e.g.,Hirschsprung's disease(HD).HD or aganglionic megacolon or aganglionosis,is a congenital defect that affects 1 in 3000～5000 newborns and it results from a congenital absence of ganglion cells(aganglionosis) in avariable length of bowel.Aganglionosis results in an uncoordinated tonic state in the affected the gut segment resulting in the clinical presentation of bowel obstruction as luminal contents fail to progress.So the affected children usually present shortly after birth with symptoms and signs of distal bowel obstruction.Symptoms range from neonatal intestinal obstruction to chronic progressive constipation in older children.Typical operative findings are of an apparently narrow distal colon/rectum and a massively dilated proximal colon.The treatment of these enteric neuromuscular disorders is far from satisfactory and remains palliative at best.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 often follows by some complications such as constipation,fecal incontinence and most serious enterocolitis and colonic rupture. The shortcomings in surgery have led to a search for new therapies to improve outcomes in HSCR.Theoretically,a real cure will restore or replace missing or dysfunctional neurons with healthy ones.This has led to the hypothesis that it may be possible to use stem cells to form a "new-ENS" in order to normalize function of the gut.Recent data showed us the exciting progress in the use of NSCs as a valuable therapeutic approach for disorders of the enteric nervous system characterized by a loss of critical neuronal subpopulations.NSCs have been identified in both the developing and adult CNS and PNS of rodents and humans.These cells can be grown in culture and display the ability to self-renew,although they are more restricted than ES cells and give rise predominantly to neurons and glia(astrocytes and oligodendrocytes).The cortex-originated neural stem cells therefore have the potential advantage as the desired phenotype.Whether NSCs could survive well and. differentiate in the gut environment has been unclear.In order to investigate NSCs as an in vitro expandable alternative source for cell replacement therapy in HD,it is essential to set up animal models which closely resemble the neuropathological characteristics of this disease.Therefore,we designed this study and the purpose of the study is to explore the potential of NSCs transplantation as a therapeutic strategy for neuronal replacement of enteric nervous system of rectum in aganglionic rat.This study is a series which consists of three parts as follows:establishment the aganglionic megacolon model by using benzalkonium chloride(BAC) to ablation the ganglionated plexuses selectly;isolation,culture,purifying,proliferation of rat NSCs in vitro;transplantation the neural stem cells into the aganglionic rectum and detection the morphological fate of the grafted cells and evaluation of the enteric motility recovery.Part One:Establishment of the animal model for experimental aganglionic megacolonThe ENS is a well-defined system of neurons and glial cells that regulates several aspects of gastrointestinal physiology.Alterations of the ENS due to disease, age,or other conditions lead to impairment of gastrointestinal function associated with serious disruptions of motility,intractable symptoms,and long-term suffering. The lack of effective therapies for these syndromes due to default of ENS has led us to explore the possibilities of novel approaches such as NSCs transplantation. Therefore,establishment an ideal,steady and repeatable aganglionosis animal model is premise of this study.According to the typical pathophysiology of aganglionosis, we applied a cationic surfactant named BAC to the serosal surface of the rat rectum to ablate the ENS selectively.Four weeks after BAC treatment,the portion treated with BAC was found to be narrowed and the proximal segment became markedly dilated. By detailed histological examination,the segment treated with BAC was confirmed to be denervated and lack of acetylcholinesterase activity;whereas normal myenteric and submucous plexuses and acetylcholinesterase activity were observed in the controls.Rectoanal inhibitory reflex(RAIR) was abolished in the treatment group.A narrow aganglionic rectum was produced successfully in the rat by topical application of BAC to ablate enteric plexus.This animal model will provide the basis for further studies on pathophysiology and new strategy of treatment of ENS disorders such as HD.Part Two:Isolation,culture,purification and proliferation of nural stem cells in vitroTo explore the characterization and the effect of transplantation of NSCs for treatment of disorders of the enteric system such as HD,we systematically investigated the isolation,culture conditions,differentiation and labeling of NSCs in vitro.The NSCs were isolated from fetal brain cortex of Wistar rats(E16) and cultured in Dulbecco's minimum essential medium(DMEM)/F12-based medium, supplemented with B27,the mitogen epidermal growth factor(EGF),and basic fibroblast growth factor(bFGF).The characterization of NSCs and differentiated cells was identified by immunocytochemistry.Their self-renewal capacity was showed by observing the formation of neurospheres and BrdU immunocytochemistry.Cells were grown as free-floating clusters(neurospheres).By 72 hours under non-adherent culture condition,dozens of NSCs had proliferated to form neurosphere-like aggregates.And after cultured for 6-7 days,neurospheres consisted of hundreds of cells developed.The generated neurosphere was characterized by nestin positive.In the presence of serum,lots of cells migrating from neurosphere differentiated into cells expressing neuronal and astrocyte markers and 40.64%of them differentiated into MAP2-positive neurons compared with 55.32%GFAP-positive astrocytes,which indicated their characteristics of stem cell.Cultures were incubated with bromodeoxyuridine(BrdU).After incorporation with BrdU,approximately 90%cells in neurosphere were detected to be BrdU-positive.The results of our study indicated that neural stem cells could be isolated from fetal brain cortex and the cultured cells maintained the characteristics of neural stem cells,including proliferation, self-renewal and multipotency.It was concluded that fetal cortex-originated NSCs might provide a powerful source of cells for experimental and clinical transplantation.Part Three:Transplantation of neural stem cells into aganglionic rectum of ratOn the basis of successful establishment an aganglionosis animal model and culture,expansion of embryonic cortex-originated neural stem cells in vitro,we explored the possibility of grafting neural stem cells into the aganglionic rectum wall as a therapeutic strategy for enteric neuronal replacement.DAPI-labeled embryonic cortex-originated neural stem cells were transplanted into the denervated rectum. After transplantation,differentiation of grafted cells was examined and the intestinal motility and reflex were assessed.Our results indicated that the grafted NSCs could survive in the gut and then they could differentiate into PGP9.5-positive neurons and GFAP-positive glial cells in vivo after 8 weeks of transplantation.Further assessment for neurochemical showed that the protein expression of neuronal nitric oxide synthase(nNOS) and choline acetyltransferase(CHAT) significantly increased. Moreover,the RAIR and electrical field stimulation(EFS)-induced response were observed in grafted group compared with no reaction in denervated group.It was concluded that cortex-originated NSCs could survive and function in the denervated rat rectum in vivo,which indicated that CNS-NSCs might provide a feasible therapeutic option for some disorders of enteric nervous system.ConclusionsIn conclusion,we set up an experimental aganglionosis animal model successfully,and also successfully isolated,cultured,purified,and expanded cortex-originated NSCs in vitro and then transplanted them into the aganglionic rectum.It showed that the engrafted NSCs could survive,differentiate into functional neuronal phenotypes and restore the defaulted function of gut.This study provided the functional evidence that transplantation of NSCs in the gut might be beneficial in the treatment of motility disorders.Although promising,these data of our study are limited in their short-term nature and further investigation are necessary to assess long-term survival of CNS-NSCs in the gut wall and their functional effect.
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