Enteric neural crest‐derived cells and neural stem cells: biology and therapeutic potential

The enteric nervous system arises from two regions of the neural crest; the vagal neural crest which gives rise to the vast majority of enteric neurones throughout the gastrointestinal tract, and the sacral neural crest which contributes a smaller number of cells that are mainly distributed within the hindgut. The migration of vagal neural crest cells into, and along the gut is promoted by GDNF, which is expressed by the gut mesenchyme and is the ligand for the Ret/GFRα1 signalling complex present on migrating vagal‐derived crest cells. Sacral neural crest cells enter the gut after it has been colonized by vagal neural crest cells, but the molecular control of sacral neural crest cell development has yet to be elucidated. Under the influence of both intrinsic and extrinsic cues, neural crest cells differentiate into glia and different types of enteric neurones at different developmental stages. Recently, the potential for neural stem cells to form an enteric nervous system has been examined, with the ultimate aim of using neural stem cells as a therapeutic strategy for some gut disorders where enteric neurones are reduced or absent.     

Enteric neural stem cell therapies for enteric neuropathies

Background

Enteric neuropathies exist as a wide range of human disorders which impact on gastrointestinal motility. Current standard therapies for enteric neuropathies are limited to surgical resection or manipulation (eg, myotomy) of affected gut segments or medical management including both therapy (eg, prokinetic pharmacotherapy) and support such as parenteral nutrition. However, such treatments often result in poor prognosis and significant morbidity. The current limitations in treatment options for enteric neuropathies underline the need for alternative approaches to treat these devastating diseases. Recent advances have highlighted the potential of enteric neural stem cells as a possible treatment option for regenerative medicine, in such cases.

Purpose

The purpose of this review is to provide an up‐to‐date synopsis of the enteric neural stem cell research field. Here, we review in detail the initial characterization of enteric neural stem cells, early preclinical studies validating their use in murine models through to the most recent findings of therapeutic rescue of diseased gut tissue. We additionally pose a number of questions regarding these recent findings which will need to be addressed prior to clinical translation of this exciting cellular therapeutic.     

Neural stem cell transplantation in the enteric nervous system: roadmaps and roadblocks

Abstract  The enteric nervous system (ENS) is vulnerable to a variety of genetic, metabolic or environmental threats, resulting in clinical disorders characterized by loss or malfunction of neuronal elements. These disorders have been difficult to treat and there is much enthusiasm for novel therapies such as neural stem cell (NSC) transplantation to restore ENS function in diseased segments of the gut. Recent research has indicated the potential for a variety of innovative approaches to this effect using NSC obtained from the central nervous system (CNS) as well as gut derived enteric neuronal progenitors. The main goal of this review is to summarize the current status of NSC research as it applies to the ENS, delineate a roadmap for effective therapeutic strategies using NSC transplantation and point out the numerous challenges that lie ahead.     

Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: requirement for a permissive microenvironment

The enteric system is formed by cells that migrate to the bowel from the neural crest. In order to gain insight into intraenteric factors that influence this migration, the colonization of the bowel of the ls/ls mouse was investigated. The terminal 2 mm of ls/ls intestine fails to become colonized by crest cells and thus remains aganglionic. The entire bowel of control mice and ls/ls mice was explanted before the appearance in situ of recognizable neurons and grown in organotypic tissue culture. Neurons, detected by the histochemical demonstration of acetylcholinesterase activity, developed throughout the length of the control gut, but, even in vitro, were excluded from the terminal segment of the ls/ls intestine. Co-culture experiments were done, in which primary and secondary sources of crest cells were combined with recipient segments of bowel, to test the ability of the recipient tissue to become colonized by neural precursors. The primary source was murine crest cells migrating away from an explant of the neuraxis. Secondary sources included avian and murine foregut (control and ls/ls) containing migratory crest cells as well as the quail ganglion of Remak. Recipient segments of bowel included control avian and murine hindgut, explanted before the tissue had become colonized by crest cells in situ, as well as the presumptive aganglionic bowel of ls/ls mice. Both primary and secondary sources of crest cells proved to be able to contribute neurons to the control segments of recipient hindgut. Species differences were no barrier to the colonization of the bowel in vitro. Moreover, the ls/ls foregut was as good a source of neural precursors for a normal recipient bowel, as was control avian or murine foregut. In contrast, none of the sources of crest cells that were utilized contributed neurons to the presumptive aganglionic gut of ls/ls mice. Both cells and processes of enteric neurons developing in vitro (detected by demonstrating neurofilament immunoreactivity) tended to be excluded from the presumptive aganglionic tissue. On the other hand, neurites, but not cell bodies, of dorsal root ganglia co-cultured with presumptive aganglionic ls/ls bowel did enter the abnormal zone. These data are consistent with the hypothesis that nonneuronal elements of the wall of the presumptive aganglionic region of the ls/ls gut are abnormal and prevent the colonization of this segment of the gut with viable neural precursors from the neural crest.(ABSTRACT TRUNCATED AT 400 WORDS)     

Advances in ontogeny of the enteric nervous system

The neurons and glia that comprise the enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract, are derived from vagal and sacral regions of the neural crest. In order to form the ENS, neural crest-derived precursors undergo a number of processes including survival, migration and proliferation, prior to differentiation into neuronal subtypes, some of which form functional connections with the gut smooth muscle. Investigation of the developmental processes that underlie ENS formation has progressed dramatically in recent years, in no small part due to the attention of scientists from a range of disciplines on the genesis of Hirschsprung's disease (aganglionic megacolon), the major congenital abnormality of the ENS. This review summarizes recent advances in the field of early ENS ontogeny and focuses on: (i) the spatiotemporal migratory pathways followed by vagal and sacral neural crest-derived ENS precursors, including recent in vivo imaging of migrating crest cells within the gut, (ii) the roles of the RET and EDNRB signalling pathways and how these pathways interact to control ENS development, and (iii) how perpendicular migrations of neural crest cells within the gut lead to the formation of the myenteric and submucosal plexi located between the smooth muscle layers of the gut wall.     

人胚神经干细胞移植治疗大鼠脑缺血的实验研究

目的　研究人胚神经干细胞(hNSCs)移植治疗脑缺血大鼠的效果及其在缺血大鼠脑内的状况。方法　从自然流产的孕10~13周的人胚脑组织中分离、培养神经干细胞。采用线栓法制作大鼠脑缺血模型,1d后经尾静脉移植未分化的hNSCs入脑缺血大鼠体内,对移植后大鼠进行神经损害严重程度评分(NSS),用免疫组化方法观察移植后hNSCs的存活、迁徙、分化状况。结果　从人胎脑中成功培养出hNSCs,培养条件下呈悬浮状态生长,形成神经球,绝大多数的细胞表达神经干细胞的标记物神经巢蛋白(nestin)。hNSCs移植组大鼠自移植后3周末起其NSS显著低于对照组(P<0 .05);移植后2、3、4、5周脑组织切片中均可见5 溴脱氧嘧啶尿苷(Brdu)染色阳性细胞,缺血侧明显多于对侧(P<0 .05),移植后3、4、5周末明显多于移植后2周(均P<0 .05);移植组各时间点脑组织切片中均可见nestin染色阳性细胞;在Brdu阳性细胞群中, 73 8%为胶质纤维酸性蛋白(GFAP)染色阳性的星形胶质细胞, 16 7%为2, 3 环核苷酸磷酸二脂酶(CNPase)染色阳性的少突胶质细胞, 9 5%为神经元特异性烯醇化酶(NSE)染色阳性的神经元。结论　经静脉移植hNSCs能有效改善脑梗死动物的神经功能,hNSCs体内体外均具有多向分化潜能,受缺血部位微环境信号的影响分化成3种主要类型的神经细胞。     

Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system

Enteric glial cells are vital for the autonomic control of gastrointestinal homeostasis by the enteric nervous system. Several different functions have been assigned to enteric glial cells but whether these are performed by specialized subtypes with a distinctive phenotype and function remains elusive. We used Mosaic Analysis with Double Markers and inducible lineage tracing to characterize the morphology and dynamic molecular marker expression of enteric GLIA in the myenteric plexus. Functional analysis in individually identified enteric glia was performed by Ca²⁺ imaging. Our experiments have identified four morphologically distinct subpopulations of enteric glia in the gastrointestinal tract of adult mice. Marker expression analysis showed that the majority of glia in the myenteric plexus co‐express glial fibrillary acidic protein (GFAP), S100β, and Sox10. However, a considerable fraction (up to 80%) of glia outside the myenteric ganglia, did not label for these markers. Lineage tracing experiments suggest that these alternative combinations of markers reflect dynamic gene regulation rather than lineage restrictions. At the functional level, the three myenteric glia subtypes can be distinguished by their differential response to adenosine triphosphate. Together, our studies reveal extensive heterogeneity and phenotypic plasticity of enteric glial cells and set a framework for further investigations aimed at deciphering their role in digestive function and disease. GLIA 2015;63:229–241     

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

Abstract  The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.     

Nestin‐expressing cells in the gut give rise to enteric neurons and glial cells

Background Neuronal stem cells (NSCs) are promising for neurointestinal disease therapy. Although NSCs have been isolated from intestinal musclularis, their presence in mucosa has not been well described. Mucosa‐derived NSCs are accessible endoscopically and could be used autologously. Brain‐derived Nestin‐positive NSCs are important in endogenous repair and plasticity. The aim was to isolate and characterize mucosa‐derived NSCs, determine their relationship to Nestin‐expressing cells and to demonstrate their capacity to produce neuroglial networks in vitro and in vivo. Methods Neurospheres were generated from periventricular brain, colonic muscularis (Musc), and mucosa–submucosa (MSM) of mice expressing green fluorescent protein (GFP) controlled by the Nestin promoter (Nestin‐GFP). Neuronal stem cells were also grown as adherent colonies from intestinal mucosal organoids. Their differentiation potential was assessed using immunohistochemistry using glial and neuronal markers. Brain and gut‐derived neurospheres were transplanted into explants of chick embryonic aneural hindgut to determine their fate. Key Results Musc‐ and MSM‐derived neurospheres expressed Nestin and gave rise to cells of neuronal, glial, and mesenchymal lineage. Although Nestin expression in tissue was mostly limited to glia co‐labelled with glial fibrillary acid protein (GFAP), neurosphere‐derived neurons and glia both expressed Nestin in vitro, suggesting that Nestin+/GFAP+ glial cells may give rise to new neurons. Moreover, following transplantation into aneural colon, brain‐ and gut‐derived NSCs were able to differentiate into neurons. Conclusions & Inferences Nestin‐expressing intestinal NSCs cells give rise to neurospheres, differentiate into neuronal, glial, and mesenchymal lineages in vitro, generate neurons in vivo and can be isolated from mucosa. Further studies are needed for exploring their potential for treating neuropathies.     

Stem cell transplantation for treatment of cerebral ischemia in rats Effects of human umbilical cord blood stem cells and human neural stem cells

BACKGROUND:Exogenous neural stem cell transplantation promotes neural regeneration. However, various types of stem cells transplantation outcomes remain controversial. OBJECTIVE:To explore distribution, proliferation and differentiation of human neural stem cells (hNSCs) and human umbilical cord blood stem cells (hUCBSCs) following transplantation in ischemic brain tissue of rats, and to compare therapeutic outcomes between hNSCs and hUCBSCs. DESIGN, TIME AND SETTING:Randomized controlled animal studies were performed at the Experimental Animal Center of Nanjing Medical University and Central Laboratory of Second Affiliated Hospital of Nanjing Medical University of China from September 2008 to April 2009. MATERIALS:hNSCs were harvested from brain tissue of 10-13 week old fetuses following spontaneous abortion, and hUCBSCs were collected from umbilical cord blood of full-term newborns at the Second Affiliated Hospital of Nanjing Medical University of China. hNSCs and hUCBSCs were labeled by 5-bromodeoxyuridine (BrdU) prior to transplantation. METHODS:Rat models of cerebral ischemia were established by the suture method. A total of 60 healthy male Sprague Dawley rats aged 7-9 weeks were randomly assigned to hNSC transplantation, hUCBSC transplantation and control groups. The rat models in the hNSC transplantation, hUCBSC transplantation and control groups were infused with hNSC suspension, hUCBSC suspension and saline via the caudal vein, respectively. MAIN OUTCOME MEASURES:The distribution, proliferation and differentiation of hNSCs and hUCBSCs in ischemic brain tissue were observed using immunohistochemical methods. Neurological function in rats was assessed using the neurological severity score. RESULTS:The number of BrdU-positive cells was significantly greater in the hNSC transplantation group compared with hUCBSC transplantation group at 14 days following transplantation (P < 0.05). The number of BrdU-positive cells reached a peak at 28 days following transplantation. Nestin-positive, glial fibrillary acidic protein-positive, cyclic nucleotide 3' phosphohydrolase-positive and neuron specific enolase-positive cells were visible following transplantation. No significant difference was determined in the constituent ratio of various cells between hNSC and hUCBSC transplantation groups (P > 0.05). The neurological severity score was significantly decreased in rats at 21 days following transplantation (P < 0.05). No significant difference was detected in neurological severity score between hNSC and hUCBSC transplantation groups at various time points (P > 0.05). CONCLUSION:The transplanted hNSCs and hUCBSCs can migrate into ischemic brain tissue, proliferate and differentiate into neuron-like, astrocyte-like and oligodendrocyte-like cells, and improve neurological function in rats with cerebral ischemia.     

In vitro differentiation of neural stem cells derived from human olfactory bulb into dopaminergic‐like neurons

This study describes a new accessible source of neuronal stem cells that can be used in Parkinson's disease cell transplant. The human olfactory bulb contains neural stem cells (NSCs) that are responsible for neurogenesis in the brain and the replacement of damaged cellular components throughout life. NSCs are capable of differentiating into neuronal and glial cells. We isolated NSCs from the olfactory bulb of brain‐death donors and differentiated them into dopaminergic neurons. The olfactory bulb tissues obtained were cultured in Dulbecco's modified Eagle's medium/nutrient mixture F12, B27 supplemented with basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor. The NSCs and proliferation markers were assessed. The multipotentiality of olfactory bulb NSCs was demonstrated by their capacity to differentiate into neurons, oligodendrocytes and astrocytes. To generate dopaminergic neurons, olfactory bulb NSCs were differentiated in neurobasal medium, supplemented with B27, and treated with sonic hedgehog, fibroblast growth factor 8 and glial cell‐derived neurotrophic factor from the 7th to the 21st day, followed by detection of dopaminergic neuronal markers including tyrosine hydroxylase and aromatic l ‐amino acid decarboxylase. The cells were expanded, established in continuous cell lines and differentiated into the two classical neuronal phenotypes. The percentage of co‐positive cells (microtubule‐associated protein 2 and tyrosine hydroxylase; aromatic l‐amino acid decarboxylase and tyrosine hydroxylase) in the treated cells was significantly higher than in the untreated cells. These results illustrate the existence of multipotent NSCs in the adult human olfactory bulb that are capable of differentiating toward putative dopaminergic neurons in the presence of trophic factors. Taken together, our data encourage further investigations of the possible use of olfactory bulb NSCs as a promising cell‐based therapeutic strategy for Parkinson's disease.     

Development of the enteric nervous system: bringing together cells, signals and genes

Abstract  The enteric nervous system (ENS), the intrinsic innervation of the gastrointestinal tract that controls essential functions such as motility, secretion and blood flow, comprises a vast number of neurons and glial cells that are organized into complex networks of interconnected ganglia distributed throughout the entire length of the gut wall. Enteric neurons and glia are derived from neural crest cells that undergo extensive migration, proliferation, differentiation and survival in order to form a functional ENS. Investigations of the developmental processes that underlie ENS formation in animal models, and of the common human congenital ENS abnormality Hirschsprung's disease, have been intimately related and recently led to major advances in the field. This review touches on some of these advances and introduces two topics that are elaborated upon in this journal issue: (i) genome wide approaches for profiling gene expression in wild type and mutant ENS that have been used to identify novel molecules with important roles in enteric neurogenesis, and (ii) the use of multilineage ENS progenitors isolated from embryonic or postnatal gut as novel cell replacement therapies for Hirschsprung's disease. Such studies will not only unravel the mechanisms underlying ENS development, but will also shed light on the pathogenesis of ENS developmental disorders and help to establish novel therapeutic strategies for restoring or repairing malfunctioning enteric neural circuits prevalent in numerous gastrointestinal diseases.     

Neuroepithelial stem cells differentiate into neuronal phenotypes and improve intestinal motility recovery after transplantation in the aganglionic colon of the rat

The purpose of this study was to elucidate the possibility and the biological significance of intracolonic grafting of neuroepithelial stem cells (NESCs) as a therapeutic strategy for neuronal replacement in disorders of the enteric nervous system (ENS) such as aganglionosis. The enteric plexus of rat colon were eliminated by serosal application of the cationic surfactant benzalkonium chloride. NESCs were harvested from the neural tube of embryonic rat, labelled with bromodeoxyuridine (BrdU), and transplanted into the denervated colon. After 2, 4 and 8 weeks, grafted cells were visualized in colon sections by fluorescent double-staining for BrdU and neuronal, astrocytic, neurochemical or stem cell markers. Eight weeks post-transplantation, the intestinal motility was assessed by measuring the changes of intraluminal pressure responding to inflating stimulation and the responses to electrical field stimulation (EFS). Our results indicate that when transplanted into the denervated gut, NESCs survived and could differentiate into neurons and glial cells in vivo. Furthermore, inflation stimulated contraction and EFS-induced response were observed in NESCs grafted group compared with no reaction in denervated group. Therefore, NESCs can survive and function in the denervated rat colon in vivo, which indicates that NESCs provide a promising cellular replacement candidate for ENS.     

Epidermal neural crest stem cells and their use in mouse models of spinal cord injury

Epidermal neural crest stem cell (EPI-NCSC) grafts cause a significant improvement in sensory connectivity and touch perception in the contused mouse spinal cord. EPI-NCSC are derived from the embryonic neural crest but reside in a postnatal location, the bulge of hair follicles. Both mouse and human EPI-NCSC are multipotent adult stem cells capable of generating all major neural crest derivatives. EPI-NCSC of mouse and human origin express the neural crest stem cell molecular signature, genes that were initially used to create induced pluripotent stem (iPS) cells, and other neural crest and global stem cell genes. Due to their origin in the neural folds and because they share a higher order stem cell, neural crest cells, and thus EPI-NCSC, are closely related to neural tube stem cells. This close ontological relationship with the spinal cord makes EPI-NCSC attractive candidates for cell-based therapy in spinal cord injury. In two different contusion models of spinal cord injury, we have shown that EPI-NCSC integrate into the murine spinal cord tissue and that subsets differentiate into GABAergic neurons and myelinating oligodendrocytes. Intraspinal EPI-NCSC do not form tumours. In the presence of EPI-NCSC grafts, but not in control animals, there is a 24% improvement of sensory connectivity and a substantial improvement in touch perception. Unilateral transplants leading to bilateral functional improvements suggest that underlying mechanisms include diffusible molecules. EPI-NCSC indeed express genes that encode neurotrophins, other trophic factors, angiogenic factors and metalloproteases. Intraspinal EPI-NCSC thus have multiple effects in the contused spinal cord, the sum of which can explain the observed functional improvements.