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.     

Endoglin is required for myogenic differentiation potential of neural crest stem cells

Genetic studies show that TGFbeta signaling is essential for vascular development, although the mechanism through which this pathway operates is incompletely understood. Here we demonstrate that the TGFbeta auxiliary coreceptor endoglin (eng, CD105) is expressed in a subset of neural crest stem cells (NCSCs) in vivo and is required for their myogenic differentiation. Overexpression of endoglin in the neural crest caused pericardial hemorrhaging, correlating with altered vascular smooth muscle cell investment in the walls of major vessels and upregulation of smooth muscle alpha-actin protein levels. Clonogenic differentiation assay of NCSCs derived from neural tube explants demonstrated that only NCSC expressing high levels of endoglin (NCSC(CD105+)) had myogenic differentiation potential. Furthermore, myogenic potential was deficient in NCSCs obtained from endoglin null embryos. Expression of endoglin in NCSCs declined with age, coinciding with a reduction in both smooth muscle differentiation potential and TGFbeta1 responsiveness. These findings demonstrate a cell autonomous role for endoglin in smooth muscle cell specification contributing to vascular integrity.     

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.     

Neural crest‐derived stem cells display a wide variety of characteristics

A recent burst of findings has shown that neural crest‐derived stem cells (NCSCs) can be found in diverse mammalian tissues. In addition to their identification in tissues that are known to be derived from the neural crest, recent studies have revealed NCSCs in tissues that are not specifically derived from the neural crest, such as bone marrow. NCSCs can express a wide range of characteristics, and which properties are expressed mainly depends on their tissue sources and the ontogenic stage of the animal. The identification of NCSCs in various tissues opens an entirely new avenue of approach to developing autologous cell replacement therapies for use in regenerative medicine. In this review, we discuss the origin, migration, and lineage potential of NCSCs from various mammalian tissue sources. J. Cell. Biochem. 107: 1046–1052, 2009. © 2009 Wiley‐Liss, Inc.     

Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells

Multipotent and self-renewing neural stem cells have been isolated in culture, but equivalent cells have not yet been prospectively identified in neural tissue. Using cell surface markers and flow cytometry, we have isolated neural crest stem cells (NCSCs) from mammalian fetal peripheral nerve. These cells are phenotypically and functionally indistinguishable from NCSCs previously isolated by culturing embryonic neural tube explants. Moreover, in vivo BrdU labeling indicates that these stem cells self-renew in vivo. NCSCs freshly isolated from nerve tissue can be directly transplanted in vivo, where they generate both neurons and glia. These data indicate that neural stem cells persist in peripheral nerve into late gestation by undergoing self-renewal. Such persistence may explain the origins of some PNS tumors in humans.     

Uniaxial mechanical strain modulates the differentiation of neural crest stem cells into smooth muscle lineage on micropatterned surfaces

Neural crest stem cells (NCSCs) play an important role in the development and represent a valuable cell source for tissue engineering. However, how mechanical factors in vivo regulate NCSC differentiation is not understood. Here NCSCs were derived from induced pluripotent stem cells and used as a model to determine whether vascular mechanical strain modulates the differentiation of NCSCs into smooth muscle (SM) lineage. NCSCs were cultured on micropatterned membranes to mimic the organization of smooth muscle cells (SMCs), and subjected to cyclic uniaxial strain. Mechanical strain enhanced NCSC proliferation and ERK2 phosphorylation. In addition, mechanical strain induced contractile marker calponin-1 within 2 days and slightly induced SM myosin within 5 days. On the other hand, mechanical strain suppressed the differentiation of NCSCs into Schwann cells. The induction of calponin-1 by mechanical strain was inhibited by neural induction medium but further enhanced by TGF-β. For NCSCs pre-treated with TGF-β, mechanical strain induced the gene expression of both calponin-1 and SM myosin. Our results demonstrated that mechanical strain regulates the differentiation of NCSCs in a manner dependent on biochemical factors and the differentiation stage of NCSCs. Understanding the mechanical regulation of NCSC differentiation will shed light on the development and remodeling of vascular tissues, and how transplanted NCSCs respond to mechanical factors.     

Neural crest-derived cells with stem cell features can be traced back to multiple lineages in the adult skin

Given their accessibility, multipotent skin-derived cells might be useful for future cell replacement therapies. We describe the isolation of multipotent stem cell-like cells from the adult trunk skin of mice and humans that express the neural crest stem cell markers p75 and Sox10 and display extensive self-renewal capacity in sphere cultures. To determine the origin of these cells, we genetically mapped the fate of neural crest cells in face and trunk skin of mouse. In whisker follicles of the face, many mesenchymal structures are neural crest derived and appear to contain cells with sphere-forming potential. In the trunk skin, however, sphere-forming neural crest-derived cells are restricted to the glial and melanocyte lineages. Thus, self-renewing cells in the adult skin can be obtained from several neural crest derivatives, and these are of distinct nature in face and trunk skin. These findings are relevant for the design of therapeutic strategies because the potential of stem and progenitor cells in vivo likely depends on their nature and origin.     

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.     

Isolation and characterization of neural crest-derived stem cells from dental pulp of neonatal mice

Dental pulp stem cells (DPSCs) are shown to reside within the tooth and play an important role in dentin regeneration. DPSCs were first isolated and characterized from human teeth and most studies have focused on using this adult stem cell for clinical applications. However, mouse DPSCs have not been well characterized and their origin(s) have not yet been elucidated. Herein we examined if murine DPSCs are neural crest derived and determined their in vitro and in vivo capacity. DPSCs from neonatal murine tooth pulp expressed embryonic stem cell and neural crest related genes, but lacked expression of mesodermal genes. Cells isolated from the Wnt1-Cre/R26R-LacZ model, a reporter of neural crest-derived tissues, indicated that DPSCs were Wnt1-marked and therefore of neural crest origin. Clonal DPSCs showed multi-differentiation in neural crest lineage for odontoblasts, chondrocytes, adipocytes, neurons, and smooth muscles. Following in vivo subcutaneous transplantation with hydroxyapatite/tricalcium phosphate, based on tissue/cell morphology and specific antibody staining, the clones differentiated into odontoblast-like cells and produced dentin-like structure. Conversely, bone marrow stromal cells (BMSCs) gave rise to osteoblast-like cells and generated bone-like structure. Interestingly, the capillary distribution in the DPSC transplants showed close proximity to odontoblasts whereas in the BMSC transplants bone condensations were distant to capillaries resembling dentinogenesis in the former vs. osteogenesis in the latter. Thus we demonstrate the existence of neural crest-derived DPSCs with differentiation capacity into cranial mesenchymal tissues and other neural crest-derived tissues. In turn, DPSCs hold promise as a source for regenerating cranial mesenchyme and other neural crest derived tissues.     

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.     

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest¹. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest^2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone^6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required¹⁰. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.Download video file.^{(56M, mov)}     

Cell surface molecules and truncal neural crest ontogeny: a perspective

The neural crest cell is synonymous with vertebrates and can be viewed as a transitory, mobile vector that conveys neuroepithelial stem cells to a diverse number of remote locations in the embryo. Neural crest cells have been studied intensively over the past 30 years, and it is increasingly apparent that their fate is, at least in part, directed extrinsically by the environment to which they are exposed in vivo. The interface between the cell surface and the opposing environment is clearly an important compartment for the correct deployment of the neural crest. Here, we review some of the molecules present in this location and how they influence the fate of the neural crest and generate disease.     

A novel model to study the dorsolateral migration of melanoblasts

Melanocytes derived from pluripotent neural crest cells migrate initially in the dorsolateral pathway between the ectoderm and dermomyotome. To understand the role of specific proteins involved in this cell migration, we looked for a cellular model that mimics the in vivo behavior of melanoblasts, and that allows functional studies of their migration. We report here that wild-type embryonic stem (ES) cells are able to follow the ventral and dorsolateral neural crest pathways after being grafted into chicken embryos. By contrast, a mutant ES cell line deficient for beta1 integrin subunits, proteins involved in cell-extracellular interactions, had a severely impaired migratory behavior. Interestingly, ES cells deficient for Kit, the tyrosine kinase receptor for the stem cell factor (SCF), behaved similarly to wild-type ES cells. Thus, grafting mouse ES cells into chicken embryos provides a new cellular system that allows both in vitro and in vivo studies of the molecular mechanisms controlling dorsolateral migration.     

Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells

Background  

Neuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously.     

Establishment by an Original Single‐cell Cloning Method and Characterization of an Immortal Mouse Melanoblast Cell Line (NCCmelb4)

We devised a unique new single‐cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20–30 cells/ml were dropped in the dish. After 1–3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single‐cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC‐S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT‐positive and tyrosinase‐negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12‐o‐tetradecanoyl‐13‐acetate (TPA) + cholera toxin (CT), the cell morphology changed and became l ‐3,4‐dihydroxyphenylalanine (DOPA)‐positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation‐inducing factors and growth factors without the effects of feeder cells.     

Isolation of a stem cell for neurons and glia from the mammalian neural crest.

We have isolated mammalian neural crest cells using a monoclonal antibody to the low affinity NGF receptor, and established conditions for the serial propagation of these cells in clonal culture to assess their developmental potential. This analysis indicates that, first, single mammalian neural crest cells are multipotent, able to generate at least neurons and Schwann cells like their avian counterparts. Second, multipotent neural crest cells generate multipotent progeny, indicating that they are capable of self-renewal and therefore are stem cells. Third, multipotent neural crest cells also generate some clonal progeny that form only neurons or glia, suggesting the production of committed neuroblasts and glioblasts. Manipulation of the substrate alters the fate of the multipotent cells. These findings have implications for models of neural crest development in vivo, and establish a system for studying the generation of cellular diversity by a multipotent stem cell in vitro.     

Effect of T3 hormone on neural differentiation of human adipose derived stem cells

Human adult stem cells, which are capable of self‐renewal and differentiation into other cell types, can be isolated from various tissues. There are no ethical and rejection problems as in the case of embryonic stem cells, so they are a promising source for cell therapy. The human body contains a great amount of adipose tissue that contains high numbers of mesenchymal stem cells. Human adipose‐derived stem cells (hADSCs) could be easily induced to form neuron‐like cells, and because of its availability and abundance, we can use it for clinical cell therapy. On the other hand, T₃ hormone as a known neurotropic factor has important impressions on the nervous system. The aim of this study was to explore the effects of T₃ treatment on neural differentiation of hADSCs. ADSCs were harvested from human adipose tissue, after neurosphere formation, and during final differentiation, treatment with T₃ was performed. Immunocytochemistry, real‐time RT‐PCR, Western blotting techniques were used for detection of nestin, MAP2, and GFAP markers in order to confirm the effects of T₃ on neural differentiation of hADSCs. Our results showed an increase in the number of glial cells but reduction in neuronal cells number fallowing T₃ treatment. Copyright © 2014 John Wiley & Sons, Ltd.     

Spatio‐temporal control of neural epithelial cell migration and epithelium‐to‐mesenchyme transition during avian neural tube development

As opposed to the neural crest, the neural epithelium is generally viewed as a static and cohesive structure. Here, using an ex vivo system free of the environmental influences and physical constraints encountered in the embryo, we show that neural epithelial cells are on the contrary intrinsically motile, although they do not undergo spontaneous epithelium‐to‐mesenchyme transition and display molecular and cellular characteristics distinct from those of neural crest cells. However, they can be instructed to undergo epithelium‐to‐mesenchyme conversion independently of the acquisition of neural crest traits. Migration potentialities of neural epithelial cells are transient and are progressively restricted during neural tube development. Restriction of cell migration is irreversible and can be in part accounted for by increase in N‐cadherin in cellular junctions and in cell polarity. In conclusion, our study reveals that the neural epithelium is a highly flexible tissue in which cells are maintained cohesive under the control of a combination of extrinsic factors and physical constraints.