Neural crest cells: From developmental biology to clinical interventions

Neural crest cells are multipotent cells, which are specified in embryonic ectoderm in the border of neural plate and epiderm during early development by interconnection of extrinsic stimuli and intrinsic factors. Neural crest cells are capable of differentiating into various somatic cell types, including melanocytes, craniofacial cartilage and bone, smooth muscle, and peripheral nervous cells, which supports their promise for cell therapy. In this work, we provide a comprehensive review of wide aspects of neural crest cells from their developmental biology to applicability in medical research. We provide a simplified model of neural crest cell development and highlight the key external stimuli and intrinsic regulators that determine the neural crest cell fate. Defects of neural crest cell development leading to several human disorders are also mentioned, with the emphasis of using human induced pluripotent stem cells to model neurocristopathic syndromes. Birth Defects Research (Part C) 102:263–274, 2014. © 2014 Wiley Periodicals, Inc.     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis

Murine neural crest stem cells (NCSCs) are a multipotent transient population of stem cells. After being formed during early embryogenesis as a consequence of neurulation at the apical neural fold, the cells rapidly disperse throughout the embryo, migrating along specific pathways and differentiating into a wide variety of cell types. In vitro the multipotency is lost rapidly, making it difficult to study differentiation potential as well as cell fate decisions. Using a transgenic mouse line, allowing for spatio-temporal control of the transforming c-myc oncogene, we derived a cell line (JoMa1), which expressed NCSC markers in a transgene-activity dependent manner. JoMa1 cells express early NCSC markers and can be instructed to differentiate into neurons, glia, smooth muscle cells, melanocytes, and also chondrocytes. A cell-line, clonally derived from JoMa1 culture, termed JoMa1.3 showed identical behavior and was studied in more detail. This system therefore represents a powerful tool to study NCSC biology and signaling pathways. We observed that when proliferative and differentiation stimuli were given, enhanced cell death could be detected, suggesting that the two signals are incompatible in the cellular context. However, the cells regain their differentiation potential after inactivation of c-MycER(T). In summary, we have established a system, which allows for the biochemical analysis of the molecular pathways governing NCSC biology. In addition, we should be able to obtain NCSC lines from crossing the c-MycER(T) mice with mice harboring mutations affecting neural crest development enabling further insight into genetic pathways controlling neural crest differentiation.     

Cranial neural crest cell migration in cockatiel Nymphicus hollandicus (Aves: Psittaciformes)

Parrots have developed unique jaw muscles in their evolutionary history. The M. pseudomasseter, which completely covers the lateral side of the jugal bar, is regarded as a jaw muscle unique to parrots. In a previous study, I presented a hypothesis on the relevance of modifications in the regulation of cranial neural crest cell (NCC) development to the generation of this novel jaw muscle based on histological analyses (Tokita [2004] J Morphol 259:69-81). In the present study, I investigated distribution and migration patterns of cranial neural crest cells (NCCs) through parrot embryogenesis with immunohistochemical techniques to further understand the role of cranial NCCs in the evolution of the M. pseudomasseter, and to provide new information on the relative plasticity in cranial NCC migration at early stages of avian development. The basic nature of cranial NCC development was mostly conserved between chick and parrot. In both, cranial NCCs migrated from the dorsal tip of the neural tube in a ventral direction. Three major populations were identified in their cranial NCCs. Migration pathways of these cells were almost identical between chick and parrot. The principal difference was seen in the relative timing of cranial NCC migration. In the parrot, cranial NCC migration into the first pharyngeal arch was more advanced than in the chick at early stages of development. Such a temporal shift in cranial NCC migration might influence architectural patterning of parrot jaw muscles that generates new muscle like M. pseudomasseter.     

Development of interstitial cells of Cajal in the human digestive tract as the result of reciprocal induction of mesenchymal and neural crest cells

Neural crest cells (NCC) can migrate into different parts of the body and express their strong inductive potential. In addition, they are multipotent and are able to differentiate into various cell types with diverse functions. In the primitive gut, NCC induce differentiation of muscular structures and interstitial cells of Cajal (ICC), and they themselves differentiate into the elements of the enteric nervous system (ENS), neurons and glial cells. ICC develop by way of mesenchymal cell differentiation in the outer parts of the primitive gut wall around the myenteric plexus (MP) ganglia, with the exception of colon, where they appear simultaneously also at the submucosal border of the circular muscular layer around the submucosal plexus (SMP) ganglia. However, in a complex process of reciprocal induction of NCC and local mesenchyma, c‐kit positive precursors are the first to differentiate, representing probably the common precursors of ICC and smooth muscle cells (SMC). C‐kit positive precursors could represent a key impact factor regarding the final differentiation of NCC into neurons and glial cells with neurons subsequently excreting stem cell factor (SCF) and other signalling molecules. Under the impact of SCF, a portion of c‐kit positive precursors lying immediately around the ganglia differentiate into ICC, while the rest differentiate into SMC.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Repulsive and attractive semaphorins cooperate to direct the navigation of cardiac neural crest cells

The cardiac neural crest, a subpopulation of the neural crest, contributes to the cardiac outflow tract formation during development. However, how it follows the defined long-range migratory pathway remains unclear. We show here that the migrating cardiac neural crest cells (NCCs) express Plexin-A2, Plexin-D1 and Neuropilin. The membrane-bound ligands for Plexin-A2, Semaphorin (Sema)6A and Sema6B, are expressed in the dorsal neural tube and the lateral pharyngeal arch mesenchyme (the NCC “routes”). Sema3C, a ligand for Plexin-D1/neuropilin-1, is expressed in the cardiac outflow tract (the NCC “target”). Sema6A and Sema6B repel neural crest cells, while Sema3C attracts neural crest cells. Sema6A and Sema6B repulsion and Sema3C attraction are diminished either when Plexin-A2 and Neuropilin-1, or when Plexin-D1, respectively, are knocked down in NCCs. When RNAi knockdown diminishes each receptor in NCCs, the NCCs fail to migrate into the cardiac outflow tract in the developing chick embryo. Furthermore, Plexin-A2-deficient mice exhibit defects of cardiac outflow tract formation. We therefore conclude that the coordination of repulsive cues provided by Sema6A/Sema6B through Plexin-A2 paired with the attractive cue by Sema3C through Plexin-D1 is required for the precise navigation of migrating cardiac NCCs.     

In the beginning: Generating neural crest cell diversity

Neural crest cells (NCCs) are migratory cells that delaminate from the neural tube early in development and then disseminate throughout the embryo to give rise to a wide variety of cell types that are key to the vertebrate body plan. During their journey from the neural tube to their peripheral targets, NCCs progressively differentiate, raising the question of when the fate of an individual NCC is sealed. One hypothesis suggests that the fate of a NCC is specified by target-derived signals emanating from the environment they migrate through, while another hypothesis proposes that NCCs are already specified to differentiate along select lineages at the time they are born in the neural tube, with environmental signals helping them to realize their prespecified fate potential. Alternatively, both mechanisms may cooperate to drive NCC diversity. This review highlights recent advances in our understanding of prespecification during trunk NCC development.Key words: neural crest cell, multipotent, prespecification, neuropilin, semaphorin, migration, cell fate     

神经嵴细胞和神经嵴病及其致病机制的研究进展

神经嵴细胞(neural crest cells,NCCs)是一类脊椎动物特有的可迁移的多能干细胞,其可分化为软骨细胞、神经元和黑色素细胞等多种类型细胞。NCCs的形成、迁移和分化受到严格调控,任何扰乱NCCs发育的因素都可导致胚胎发育畸形。由神经嵴细胞发育异常所导致的一系列疾病统称为神经嵴病(neurocristopathies,NCPs)。NCPs种类繁多且表型复杂,可累及人体多个部位(颅面部、心脏、肠胃和皮肤等),严重危害患者的身体机能和心理健康。NCPs占所有出生缺陷患儿的1/3,遗传因素是导致NCPs的主要风险因素,但环境风险因子以及基因–环境交互作用异常也可导致NCPs。本文对神经嵴细胞和神经嵴病及其致病机制进行综述,为系统认知神经嵴细胞发育以及神经嵴病提供参考,为了解神经嵴病的病因以及开展有效防控提供科学支撑。     

Human epidermal neural crest stem cells as candidates for cell‐based therapies,disease modeling,and drug discovery

In this review article I explore the suitability of human epidermal neural crest stem cells (hEPI‐NCSC) for translational medicine. hEPI‐NCSC are multipotent somatic stem cells that are derived from the embryonic neural crest. hEPI‐NCSC are located in the bulge of hair follicles where they persist postnatally and into adulthood. Because of their location in the hairy skin and their migratory behavior, hEPI‐NCSC can be easily isolated as a highly pure population of stem cells without the need for purification. Furthermore they can be expanded ex vivo into millions of stem cells, they do not form tumors in vivo, and they can undergo directed differentiation into crest and noncrest‐derived cell types of clinical relevance. Taken together, these characteristics make hEPI‐NCSC attractive candidates for cell‐based therapies, drug discovery, and disease modeling. Birth Defects Research (Part C) 102:221–226, 2014. © 2014 Wiley Periodicals, Inc.     

Peripheral sensory neurons differentiate from neural precursors derived from human embryonic stem cells

Abstract Neural precursors have been derived from human embryonic stem cells (hESC) using the bone morphogenetic protein antagonist noggin. These neural precursors can be further differentiated to produce neural cells that express central nervous system (CNS) markers. We have recently shown that naïve hESC can be directed to differentiate into peripheral sensory (PS) neuron-like cells and putative neural crest precursors by co-culturing with PA6 stromal cells. In the present study, we examine whether hESC-derived neural precursors (NPC) can differentiate into the peripheral nervous system, as well as CNS cells. As little as 1 week after co-culture with PA6 cells, cells with the molecular characteristics of PS neurons and neural crest are observed in the cultures. With increased time in culture, more PS-like neurons appear, in parallel with a reduction in the neural crest-like cells. These results provide the first evidence that neural precursors derived from hESC have the potential to develop into PS neurons-like as well as CNS-like neuronal cells. About 10% of the cells in NPC-PA6 co-cultures express PS neuron markers after 3 weeks, compared with <1% of hESC cultured on PA6. This enrichment for peripheral neurons makes this an attractive system for generation of peripheral neurons for pathophysiology study and drug development for diseases of the peripheral nervous system such as Familial Dysautonomia and varicella virus infection.     

In the beginning

Neural crest cells (NCCs) are migratory cells that delaminate from the neural tube early in development and then disseminate throughout the embryo to give rise to a wide variety of cell types that are key to the vertebrate body plan. During their journey from the neural tube to their peripheral targets, NCCs progressively differentiate, raising the question when the fate of an individual NCC is sealed. One hypothesis suggests that the fate of a NCC is specified by target-derived signals emanating from the environment they migrate through, while another hypothesis proposes that NCCs are already specified to differentiate along select lineages at the time they are born in the neural tube, with environmental signals helping them to realize their prespecified fate potential. Alternatively, both mechanisms may cooperate to drive NCC diversity. This review highlights recent advances in our understanding of prespecification during trunk NCC development.     

Neural crest derivatives in ocular development: Discerning the eye of the storm

Neural crest cells (NCCs) are vertebrate‐specific transient, multipotent, migratory stem cells that play a crucial role in many aspects of embryonic development. These cells emerge from the dorsal neural tube and subsequently migrate to different regions of the body, contributing to the formation of diverse cell lineages and structures, including much of the peripheral nervous system, craniofacial skeleton, smooth muscle, skin pigmentation, and multiple ocular and periocular structures. Indeed, abnormalities in neural crest development cause craniofacial defects and ocular anomalies, such as Axenfeld‐Rieger syndrome and primary congenital glaucoma. Thus, understanding the molecular regulation of neural crest development is important to enhance our knowledge of the basis for congenital eye diseases, reflecting the contributions of these progenitors to multiple cell lineages. Particularly, understanding the underpinnings of neural crest formation will help to discern the complexities of eye development, as these NCCs are involved in every aspect of this process. In this review, we summarize the role of ocular NCCs in eye development, particularly focusing on congenital eye diseases associated with anterior segment defects and the interplay between three prominent molecules, PITX2, CYP1B1, and retinoic acid, which act in concert to specify a population of neural crest‐derived mesenchymal progenitors for migration and differentiation, to give rise to distinct anterior segment tissues. We also describe recent findings implicating this stem cell population in ocular coloboma formation, and introduce recent evidence suggesting the involvement of NCCs in optic fissure closure and vascular development. Birth Defects Research (Part C) 105:87–95, 2015. © 2015 Wiley Periodicals, Inc.     

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition ^1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells ^1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components ³. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration ⁴. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands ^5-8. However, not until recently have any chemoattractants of trunk NCCs been identified ⁹. Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere¹⁰). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.     

Dual origin of melanocytes defined by Sox1 expression and their region‐specific distribution in mammalian skin

Melanocytes are pigment‐producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1⁺ dorsal neuroepithelial cells but are derived from Sox1^? cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1^? cells clearly segregate from cells that originated from Sox1⁺ dorsal neuroepithelial cell‐derived NCCs. The possible derivation of Sox1^? cells from epidermal cells also strengthens their non‐neuroepithelial origin.     

The neural crest,A multifaceted structure of the vertebrates

In this review, several features of the cells originating from the lateral borders of the primitive neural anlagen, the neural crest (NC) are considered. Among them, their multipotentiality, which together with their migratory properties, leads them to colonize the developing body and to participate in the development of many tissues and organs. The in vitro analysis of the developmental capacities of single NC cells (NCC) showed that they present several analogies with the hematopoietic cells whose differentiation involves the activity of stem cells endowed with different arrays of developmental potentialities. The permanence of such NC stem cells in the adult organism raises the problem of their role at that stage of life. The NC has appeared during evolution in the vertebrate phylum and is absent in their Protocordates ancestors. The major role of the NCC in the development of the vertebrate head points to a critical role for this structure in the remarkable diversification and radiation of this group of animals. Birth Defects Research (Part C) 102:187–209, 2014. © 2014 Wiley Periodicals, Inc.     

Semaphorin signaling guides cranial neural crest cell migration in zebrafish

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We find that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs.     

In Vivo Expression of EphrinA5-Fc in Mice Results in Cephalic Neural Crest Agenesis and Craniofacial Abnormalities

Eph receptors and their ligands ephrins have been implicated in guiding the directed migration of neural crest cells (NCCs). In this study, we found that Wnt1-Cre-mediated expression of ephrinA5-Fc along the dorsal midline of the dien- and mesencephalon resulted in severe craniofacial malformation of mouse embryo. Interestingly, expression of cephalic NCC markers decreased significantly in the frontonasal process and branchial arches 1 and 2, which are target areas for the migratory cephalic NCCs originating in the dien- and mesencephalon. In addition, these craniofacial tissues were much smaller in mutant embryos expressing ephrinA5-Fc. Importantly, EphA7-positive cephalic NCCs were absent along the dorsal dien- and mesencephalon of mutant embryos expressing ephrinA5-Fc, suggesting that the generation of cephalic NCCs is disrupted due to ephrinA5-Fc expression. NCC explant experiments suggested that ephrinA5-Fc perturbed survival of cephalic NCC precursors in the dorsal midline tissue rather than affecting their migratory capacity, which was consistent with our previous report that expression of ephrinA5-Fc in the dorsal midline is responsible for severe neuroepithelial cell apoptotic death. Taken together, our findings strongly suggest that expression of ephrinA5-Fc decreases a population of cephalic NCC precursors in the dorsal midline of the dien- and mesencephalon, thereby disrupting craniofacial development in the mouse embryos.