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.     

In vivo transplantation of fetal human gut‐derived enteric neural crest cells

The prospect of using neural cell replacement for the treatment of severe enteric neuropathies has seen significant progress in the last decade. The ability to harvest and transplant enteric neural crest cells (ENCCs) that functionally integrate within recipient intestine has recently been confirmed by in vivo murine studies. Although similar cells can be harvested from human fetal and postnatal gut, no studies have as yet verified their functional viability upon in vivo transplantation. We sought to determine whether ENCCs harvested from human fetal bowel are capable of engraftment and functional integration within recipient intestine following in vivo transplantation into postnatal murine colon. Enteric neural crest cells selected and harvested from fetal human gut using the neurotrophin receptor p75^NTR were lentivirally labeled with either GFP or calcium‐sensitive GCaMP and transplanted into the hindgut of Rag2^?/γc^?/C5^?‐immunodeficient mice at postnatal day 21. Transplanted intestines were assessed immunohistochemically for engraftment and differentiation of donor cells. Functional viability and integration with host neuromusculature was assessed using calcium imaging. Transplanted human fetal gut‐derived ENCC showed engraftment within the recipient postnatal colon in 8/15 mice (53.3%). At 4 weeks posttransplantation, donor cells had spread from the site of transplantation and extended projections over distances of 1.2 ± 0.6 mm (n = 5), and differentiated into enteric nervous system (ENS) appropriate neurons and glia. These cells formed branching networks located with the myenteric plexus. Calcium transients (change in intensity F/F0 = 1.25 ± 0.03; 15 cells) were recorded in transplanted cells upon stimulation of the recipient endogenous ENS demonstrating their viability and establishment of functional connections.     

The Enteric Glia: Identity and Functions

Enteric glial cells were first described at the end of the 19th century, but they attracted more interest from researchers only in the last decades of the 20th. Although, they have a different embryological origin, the enteric GLIA share many characteristics with astrocytes, the main glial cell type of the central nervous system (CNS), such as in their expression of the same markers and in their functions. Here we review the construction of the enteric nervous system (ENS), with a focus on enteric glia, and also the main studies that have revealed the action of enteric glia in different aspects of gastrointestinal tract homeostasis, such as in the intestinal barrier, in communications with neurons, and in their action as progenitor cells. We also discuss recent discoveries about the roles of enteric glia in different disorders that affect the ENS, such as degenerative pathologies including Parkinson's and prion diseases, and in cases of intestinal diseases and injury. GLIA 2015;63:921–935     

Caspase inhibition increases survival of neural stem cells in the gastrointestinal tract

Neural stem cell (NSC) transplantation is a promising tool for the restoration of the enteric nervous system in a variety of motility disorders. Post-transplant survival represents a critical limiting factor for successful repopulation. The aim of this study was to determine the role of both immunological as well as non-immune-mediated mechanisms on post-transplant survival of NSC in the gut. Mouse CNS-derived NSC (CNS-NSC) were transplanted into the pylorus of recipient mice with and without the addition of a caspase-1 inhibitor (Ac-YVAD-cmk) in the injection media. In a separate experiment, CNS-NSC were transplanted in the pylorus of mice that were immunosuppressed by administration of cyclosporin A (CsA). Apoptosis and proliferation of the implanted cells was assessed 1 and 7 days post-transplantation. Survival was assessed 1 week post-transplantation. The degree of immunoresponse was also measured. The addition of a caspase-1 inhibitor significantly reduced apoptosis, increased proliferation and enhanced survival of CNS-NSC. CsA-treatment did not result in improved survival. Our results indicate that caspase-1 inhibition, but not immunosuppression, improves survival of CNS-NSC in the gut. Pre-treatment with a caspase-1 inhibitor may be a practical method to enhance the ability of transplanted CNS-NSC to survive in their new environment.     

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)     

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.     

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.     

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.     

Human neural stem cells genetically modified for brain repair in neurological disorders

Existence of multipotent neural stem cells (NSC) has been known in developing or adult mammalian CNS, including humans. NSC have the capacity to grow indefinitely and have multipotent potential to differentiate into three major cell types of CNS, neurons, astrocytes and oligodendrocytes. Stable clonal lines of human NSC have recently been generated from the human fetal telencephalon using a retroviral vector encoding v‐myc. One of the NSC lines, HB1.F3, carries normal human karyotype of 46XX and has the ability to self‐renew, differentiate into cells of neuronal and glial lineages, and integrate into the damaged CNS loci upon transplantation into the brain of animal models of Parkinson disease, HD, stroke and mucopolysaccharidosis. F3 human NSC were genetically engineered to produce L‐dihydroxyphenylalanine (L‐DOPA) by double transfection with cDNA for tyrosine hydroxylase and guanosine triphosphate cylohydrolase‐1, and transplantation of these cells in the brain of Parkinson disease model rats led to L‐DOPA production and functional recovery. Proactively transplanted F3 human NSC in rat striatum, supported the survival of host striatal neurons against neuronal injury caused by 3‐nitropro‐pionic acid in rat model of HD. Intravenously introduced through the tail vein, F3 human NSC were found to migrate into ischemic lesion sites, differentiate into neurons and glial cells, and improve functional deficits in rat stroke models. These results indicate that human NSC should be an ideal vehicle for cell replacement and gene transfer therapy for patients with neurological diseases. In addition to immortalized human NSC, immortalized human bone marrow mesenchymal stem cell lines have been generated from human embryonic bone marrow tissues with retroviral vectors encording v‐myc or teromerase gene. These immortalized cell lines of human bone marrow mesenchymal stem cells differentiated into neurons/glial cells, bone, cartilage and adipose tissue when they were grown in selective inducing media. There is further need for investigation into the neurogenic potential of the human bone marrow stem cell lines and their utility in animal models of neurological diseases.     

Vasopressin cells in the medial amygdala of the rat project to the lateral septum and ventral hippocampus

The rat brain contains a large number of vasopressin (VP) immunoreactive fibers, the sites of origin of which have not yet been established completely. For instance, the sources of VP fiber systems in the amygdala, ventral hippocampus (VH), mediodorsal thalamic nucleus, ventral tegmental area, and dorsal raphe yet remain obscure. These VP fibers may originate in any of the recently described extrahypothalamic VP cell groups, viz., medial amygdaloid nucleus (AME), dorsomedial hypothalamic nucleus, or locus coeruleus, since VP efferents from these cells still remain to be demonstrated. In search of AME VP efferents three approaches were followed: (1) the Phaseolus vulgaris anterograde tracing method, (2) immunocytochemistry after AME lesioning, and (3) retrograde transport of a fluorescent dye in combination with immunofluorescence. The results demonstrate that VP cells in the AME project to (1) the lateral septum (LS) by the ventral amygdalofugal pathway and (2) the VH via the amygdalohippocampal transition zone. In addition, the VP projection from the bed nucleus of the stria terminalis (BST) to the LS was confirmed. There was no indication that VP cells in the AME project through the amygdalotegmental pathway to the medulla oblongata and spinal cord. The results support the possibility that the BST and AME are an anatomical entity that may be part of the central loci controlling sexual processes in the rat.     

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.     

Derivation of primitive neural stem cells from human-induced pluripotent stem cells

Human-induced pluripotent stem cells (hiPSCs) have facilitated studies on organ development and differentiation into specific lineages in in vitro systems. Although numerous studies have focused on cellular differentiation into neural lineage using hPSCs, most studies have initially evaluated embryoid body (EB) formation, eventually yielding terminally differentiated neurons with limited proliferation potential. This study aimed to establish human primitive neural stem cells (pNSCs) from exogene-free hiPSCs without EB formation. To derive pNSCs, we optimized N2B27 neural differentiation medium through supplementation of two inhibitors, CHIR99021 (GSK-3 inhibitor) and PD0325901 (MEK inhibitor), and growth factors including basic fibroblast growth factor (bFGF) and human leukemia inhibitory factor (hLIF). Consequently, pNSCs were efficiently derived and cultured over a long term. pNSCs displayed differentiation potential into neurons, astrocytes, and oligodendrocytes. These early NSC types potentially promote the clinical application of hiPSCs to cure human neurological disorders.     

神经干细胞的来源

神经干细胞是近年来神经科学领域研究的一个热点。神经干细胞可来源于胚胎干细胞和成年干细胞，前者包括早期胚胎细胞和胎儿神经组织细胞，由于从胚胎获取干细胞面l临伦理学的束缚，从成年来源的神经干细胞将是未来临床应用更具可行性的途径。成年来源的神经干细胞包括存在于成年神经组织中的干细胞和从其他组织中分化得到的干细胞，其中骨髓基质细胞具有多分化潜能，在适当的条件下可以诱导分化出神经干细胞，目前备受关注。     

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     

Expression of a neurally related laminin binding protein by neural crest-derived cells that colonize the gut: relationship to the formation of enteric ganglia.

In order to give rise to the enteric nervous system (ENS), cells migrating from the neural crest must find the bowel and cease migrating at appropriate locations within the gut. Previous studies of the development of the ENS in a mutant mouse have led to the hypothesis that laminin in the enteric mesenchyme may act as a signal to crest-derived cells to cease migrating and extend neurites (or glial processes). Implied in this hypothesis is the idea that crest-derived cells, as a prelude to their participation in ganglion formation, acquire a neurally related laminin receptor, which they do not express at pre-enteric stages of migration. As a partial test of this hypothesis, single and double label immunocytochemistry at light and electron microscopic (EM) levels were used to study the expression of cell surface laminin binding proteins by crest-derived cells in the process of migrating to or within the developing chick gut. Two antibodies (called 3070 and alpha-110) raised against neuronal cell surface laminin binding proteins were employed for this purpose. Laminin binding protein immunoreactivity was found to be expressed within the bowel and ganglion of Remak by a subset of crest-derived cells (identified immunocytochemically with NC-1/HNK-1 antibodies) and by all of those developing as neurons (identified immunocytochemically with antibodies to neurofilament-associated proteins). Laminin binding protein immunoreactivity was also found to be expressed in fixed neural structures elsewhere in the embryos, including cranial and spinal roots, nerves, and ganglia. In contrast, laminin binding protein immunoreactivity was not expressed by migrating crest-derived cells in the vicinity of the vagal or sacral regions of the neuraxis (from which the precursors of the ENS take origin); nor was it expressed by juxta-pharyngeal vagal crest-derived cells migrating to the foregut through the caudal branchial arches or by the caudal stream of sacral crest-derived cells approaching the hindgut. EM immunocytochemistry confirmed that laminin binding protein immunoreactivity in the bowel was located on the surfaces of crest-derived cells, and was exhibited both by those cells that could only be distinguished from their neighbors by their NC-1/HNK-1 immunoreactivity and by cells developing as neurons or glia. EM immunocytochemistry also revealed that the surfaces of crest-derived cells migrating through the enteric mesenchyme were contacted by many small osmiophilic "puffs" of laminin-immunoreactive extracellular material. These puffs coincided in location with membrane sites that expressed the immunoreactivity of the laminin binding protein. These observations are consistent with the hypothesis that laminin plays a role in the formation of enteric ganglia.     

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.