White matter repair: skin-derived precursors as a source of myelinating cells

Stem cell based therapies hold great promise for repair and functional restoration following neurological injury and disease. Skin-derived precursors (or "SKPs") are a novel, multipotent somatic stem cell that resides within the mammalian dermis. SKPs persist within the skin throughout adulthood and yet intriguingly, exhibit many similarities to embryonic neural crest stem cells (NCSCs). For example, SKPs give rise to both neural and mesodermal cell types, and the former appear biased to peripheral nervous system fates. As such, SKPs are capable of generating Schwann cells, the myelinating glial cell of the peripheral nervous system. Here we discuss our current understanding of the biological origin of SKPs and specifically the potential therapeutic utility of SKPs as a highly accessible and autologous source of Schwann cells for remyelination and repair of the injured or diseased nervous system.     

Astrocytic and neuronal fate of mesenchymal stem cells expressing nestin

Classically, bone marrow mesenchymal stem cells (MSC) differentiate in vivo or in vitro into osteocytes, chondrocytes, fibroblasts and adipocytes. Recently, it was reported by several groups that MSC can also adopt a neural fate in appropriate in vivo or in vitro experimental conditions. However, it is unclear if those cells are really able to differentiate into functional neural cells and in particular into functional neurons. Some observations suggest that a cell fusion process underlies the neural fate adoption by MSC in vivo and first attempts to reproduce in vitro this neural fate decision in MSC cultures were unsuccessful. More recently, however, in several laboratories including ours, differentiation of MSC cultivated from adult rat bone marrow into astrocytes and neuron-like cells was demonstrated. More precisely, we stressed the importance of the expression by MSC of nestin, an intermediate filament protein associated with immaturity in the nervous system, as a pre-requisite to adopting an astrocytic or a neuronal fate in a co-culture paradigm. Using this approach, we have also demonstrated that the MSC-derived neuron-like cells exhibit several electrophysiological key properties classically devoted to neurons, including firing of action potentials. In this review, we will discuss the neurogenic potential of MSC, the factor(s) required for such plasticity, the molecular mechanism(s) underlying this neural plasticity, the importance of the environment of MSC to adopt this neural fate and the therapeutic potential of these observations.     

Differentiation of autonomic neuron precursors in vitro: cholinergic and adrenergic traits in cultured neural crest cells

The development of autonomic neuronal precursors was studied in cultures of microsurgically excised quail neural crest grown alone and associated with other young embryonic tissues. Biochemical differentiation in the cultures was followed by measuring their ability to synthesize acetylcholine (ACh) and catecholamines (CA) from radioactive precursors; cytochemical aspects of their differentiation were examined by techniques including electron microscopy, cholinesterase histochemistry, and CA cytofluorescence. Mesencephalic crest, which can make ACh before explantation, always synthesized ACh after 7 d in culture and often, but not invariably, elaborated small quantities of CA as well. Association with 2-d somite and notochord, 2-d heart, or 4-d hindgut, in medium supplemented with horse serum, resulted in the synthesis of increased amounts of both transmitters. ACh-synthesizing activity was lower and the cholinergic-stimulating effects of somite and heart were abolished in the presence of fetal calf serum. Cervicothoracic (trunk) crest, taken from the level where the dorsal mesoderm is still unsegmented, always produced ACh after culture, but CA was detectable only when the cultures were obtained by initially explanting the entire neural primordium. Co-culture of trunk crest with young embryonic tissue increased ACh-synthesizing ability and initiated CA production. Despite their capacity to elaborate neurotransmitter, cultures of either type of neural crest, alone or in association with the above-mentioned tissues, contained very few cells resembling neurons in their phase contrast appearance and none that reacted positively to any of the cytological tests applied. On the other hand, when the sclerotomic moiety of 3-d somite was cultured, trunk neural crest cells that had already migrated into the rudiment in vivo but which had not yet begun to produce detectable amounts of CA underwent rapid differentiation into neurons that synthesized and accumulated large quantities of CA. Stores of CA were detectable cytochemically as early as 24 hr after explantation and the presence of many small, dense core vesicles in neurons and processes was revealed by electron microscopy. ACh-synthesizing activity, demonstrable in freshly dissected sclerotomes, was also present in all of the cultures examined. These results show that (1) during ontogeny, cholinergic traits appear earlier than adrenergic ones in the neuronal precursors contained in the neural crest; (2) some decisive step in the differentiation of the precursor cells of the sympathetic ganglia takes place in vivo within a few hours of the onset of trunk neural crest migration. This coincides with a maturation of the somitic mesenchyme. A similar developmental process does not occur in vitro when 2-d somites and neural crest are associated in histiotypic cultures.     

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.     

Migration and differentiation of neural crest and ventral neural tube cells in vitro: implications for in vitro and in vivo studies of the neural crest

During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.     

The formation of the cranial ganglia by placodally-derived sensory neuronal precursors

The generation of the sensory ganglia involves the migration of a precursor population to the site of ganglion formation and the differentiation of sensory neurons. There is, however, a significant difference between the ganglia of the head and trunk in that while all of the sensory neurons of the trunk are derived from the neural crest, the majority of cranial sensory neurons are generated by the neurogenic placodes. In this study, we have detailed the route through which the placodally-derived sensory neurons are generated, and we find a number of important differences between the head and trunk. Although, the neurogenic placodes release neuroblasts that migrate internally to the site of ganglion formation, we find that there are no placodally-derived progenitor cells within the forming ganglia. The cells released by the placodes differentiate during migration and contribute to the cranial ganglia as post-mitotic neurons. In the trunk, it has been shown that progenitor cells persist in the forming Dorsal Root Ganglia and that much of the process of sensory neuronal differentiation occurs within the ganglion. We also find that the period over which neuronal cells delaminate from the placodes is significantly longer than the time frame over which neural crest cells populate the DRGs. We further show that placodal sensory neuronal differentiation can occur in the absence of local cues. Finally, we find that, in contrast to neural crest cells, the different mature neurogenic placodes seem to lack plasticity. Nodose neuroblasts cannot be diverted to form trigeminal neurons and vice versa.     

Derivation of neural precursors from human embryonic stem cells in the presence of noggin

The utilization of human embryonic stem cells (hESC) for basic and applied research is hampered by limitations in directing their differentiation. Empirical poorly defined methods are currently used to develop cultures enriched for distinct cell types. Here, we report the derivation of neural precursors (NPs) from hESC in a defined culture system that includes the bone morphogenetic protein antagonist noggin. When hESC are cultured as floating aggregates in defined medium and BMP signaling is repressed by noggin, non-neural differentiation is suppressed, and the cell aggregates develop into spheres highly enriched for proliferating NPs. The NPs can differentiate into astrocytes, oligodendrocytes, and mature electrophysiologically functional neurons. During prolonged propagation, the differentiation potential of the NPs shifts from neuronal to glial fate. The presented noggin-dependent controlled conversion of hESC into NPs is valuable for the study of human neurogenesis, the development of new drugs, and is an important step towards the potential utilization of hESC in neural transplantation therapy.     

A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth

Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF β-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.     

Synthesis an accumulation of putative neurotransmitters by cultured neural crest cells

The events mediating the differentiation of embryonic neural crest cells into several types of neurons are incompletely understood. In order to probe one aspect of this differentiation, we have examined the capacity of cultured quail trunk neural crest cells to synthesize, from radioactive precursors, and store several putative neurotransmitter compounds. These neural crest cultures develop the capacity to synthesize and accumulate acetylcholine and the catecholamines norepinephrine and dopamine. In contrast, detectable but relatively little synthesis and accumulation of 5-hydroxytryptamine gamma-aminobutyric acid, or octopamine from the appropriate radiolabeled precursors were observed. The capacity for synthesis and accumulation of radiolabeled acetylcholine and catecholamines is very low or absent at 2 days in vitro. Between 3 and 7 days in vitro, there is a marked rise in both catecholamine and acetylcholine accumulation in the cultures. These findings suggest that, under the particular conditions used in these experiments, the development of neurotransmitter biosynthesis in trunk neural crest cells ijs restricted and resembles, at least partially, the pattern observed in vivo. The development of this capacity to synthesize and store radiolabeled acetylcholine and catecholamines from the appropriate radioactive precursors coincides closely with the development of the activities of the synthetic enzymes choline acetyltransferase and dopamine beta-hydroxylase reported by others.     

Stage-specific and opposing roles of BDNF, NT-3 and bFGF in differentiation of purified callosal projection neurons toward cellular repair of complex circuitry

Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry.     

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.     

Morphogenesis of adrenergic cells in a frog parasympathetic ganglion

The development of the parasympathetic cardiac ganglion of the frog Xenopus laevis was marked by the differentiation of a population of adrenergic small intensely fluorescent (SIF) cells. The neural crest contributes precursors for both SIF cells and the well-studied cholinergic cardiac ganglion neurons; this situation provided an opportunity to determine whether morphogenesis of the two cell types was correlated. Accordingly, we examined the initial differentiation, developmental regulation, and territorial domain of cardiac SIF cells for comparison with their cholinergic neuron neighbors. Adrenergic SIF cells were present during the time when the first cholinergic precursors were becoming postmitotic. Although SIF cells were present first, cholinergic neurons differentiated almost 16 times faster during the first week of embryonic and larval development and outnumbered SIF cells at all subsequent stages. Nonetheless, the accumulation of both cell types were correlated, since the ratio of cholinergic neurons to SIF cells remained at approximately 10 to 1 up to adult life. Early in development, SIF cells and cholinergic neurons were clustered in the sinus venosus portion of the atrium. The asymmetric distribution of cholinergic neurons within the atrium was lost but that of the SIF cells was maintained throughout life. These results identify relationships between the morphogenesis of two cell types in an autonomic ganglion and place constraints upon the cellular mechanisms that could produce the cells from their neural crest precursors.     

Embryonic cerebral cortex cells retain CNS phenotypes after transplantation into peripheral nerve

Differentiation of stem cells depends on environmental cues. In this study, acutely dissociated or expanded cells derived from embryonic day 14 (E14) rat cerebral cortex were transplanted into the distal tibial nerve stump of adult Fischer rats to determine whether a peripheral nervous system (PNS) environment would influence cell differentiation. Acutely dissociated cells, which included neural precursors and post-mitotic neurons, were transplanted immediately after harvest. Expanded cortical cells were transplanted after 8 days of culture with fibroblast growth factor-2 (FGF-2), a process that yields a population of neural stem cells and/or neural precursors. After 2 or 10 weeks in peripheral nerve, the majority of the transplanted cells was astrocytes, as judged from glial fibrillary acid protein (GFAP) expression. Only acutely dissociated transplants had cells that exhibited neuronal phenotypes. Those neurons present in transplants at 10 weeks stained positive for glutamate decarboxylase and did not reinnervate muscle. Maintenance of this cortical phenotype in peripheral nerve suggests that it is necessary to transplant cells with neural phenotypes appropriate for muscle to restore its function.     

Differentiation of neurogenic precursors within the neural crest cell lineage

It has been suggested that many, if not all crest-derived neurons develop from a limited subpopulation of neurogenic precursors. To develop cell-type specific markers that identify these precursors directly we have used differential screening of crest-derived cell populations known to have, or not to have, neurogenic ability. We have determined that the neuronspecific human auto-antibodies designated Anti-Hu bind to cytoplasmic and nuclear determinants not only in mature avian neurons and neuroendocrine cells but also in subpopulations of morphologically non-neuronal avian crest-derived cells. Significantly, these Anti-Hu⁺ non-neuronal crest-derived cells are present only in populations that have neurogenic ability and are absent from populations that lack neurogenic ability. Moreover, following additional development in vivo or in vitro, Anti-Hu⁺ non-neuronal crest-derived cells appear to express other neuronal traits. These results suggest that Anti-Hu-immunoreactivity is an early indicator of neurogenesis among crest-derived cells and that Anti-Hu⁺ non-neuronal cells are either neurogenic precursors or immature neurons. Similarly, using the same differential screening paradigm, we have identified two monoclonal antibodies, designated 12E10 and 17F5, which also label both neurons and some apparently nonneuronal cells in neurogenic populations of neural crest cells. Anti-Hu-IR appears to precede expression of either of these two markers.     

Retinoic acid induction of ES-cell-derived neurons: the radial glia connection

Neuronal induction by retinoic acid (RA) is commonly used in embryonic stem (ES) cell differentiation. Two recent papers show that this paradigm induces a population of neurogenic precursors with properties of radial glia. Upon differentiation, RA-treated cells give rise to a defined and developmentally restricted neuronal lineage. This role of RA in cell fate specification provides new perspectives for studying the radial glia-neuron transition and for generating homogenous populations of neurons from ES cells.     

Neural precursor differentiation following transplantation into neocortex is dependent on intrinsic developmental state and receptor competence.

Reconstruction of neocortical circuitry by transplantation of neural precursors, or by manipulation of endogenous precursors, may depend critically upon both local microenvironmental control signals and the intrinsic competence of populations of precursors to appropriately respond to external molecular controls. Dependence on the developmental state of donor or endogenous precursor cells in achieving appropriate differentiation, integration, and connectivity is not clearly understood. Recent studies have demonstrated the ability to generate expandable, often clonal neural precursors at various stages of development. Transplantation of a variety of these precursors suggests that precursor differentiation and integration within the central nervous system (CNS) may depend directly on the level of cellular maturation, with less differentiated, earlier stage precursors offering more flexible but less efficient integration and more differentiated, later stage precursors offering more efficient differentiation to specific phenotypes. To further investigate this hypothesis within neocortex, we used the relatively immature HiB5 multipotent neural precursor cell line derived from embryonic day 16 hippocampus, which is less mature than precursor types that have demonstrated neuronal differentiation in adult neocortex. HiB5 cells labeled fluorescently, radioactively, and genetically were transplanted into murine neocortex under three different conditions expected to offer varying levels of instructive and permissive microenvironmental signals: (1) the developing cortex in utero; (2) regions of adult neocortex undergoing targeted pyramidal neuronal degeneration in which developmental signals are upregulated and in which later stage precursors and immature neurons undergo directed pyramidal neuron differentiation; or (3) the intact adult neocortex. Differentiation and integration of transplanted cells were examined histologically and immunocytochemically by morphology and using neuronal- and glial-specific markers. We found that these precursors underwent differentiation toward cortical neuron phenotypes with characteristic morphologies when transplanted in utero, but failed to do so under either of the adult conditions. HiB5 precursors demonstrated highly immature characteristics in vitro, consistently expressing neuroepithelial but not glial or neuronal markers. Under all conditions, donor cells survived and migrated 1-2 mm from the injection track 2 to 4 weeks after transplantation. HiB5 neural precursors transplanted into the developing cortex of embryonic mice in utero migrated within the cortex, integrated well into the host parenchyma, and differentiated toward morphologically diverse, neuronal phenotypes. HiB5 cells transplanted into the intact cortex of adult mice survived, but did not show neuronal differentiation. In contrast to slightly later stage neural precursors and embryonic neurons used in previous transplantation studies, the HiB5 cells also failed to undergo neuronal differentiation after transplantation into regions undergoing induced apoptotic neuronal degeneration in adult cortex. These results suggested that these early hippocampal-derived precursors might not be fully competent to respond to later stage differentiation and/or survival signals important in neocortex and known to be upregulated in regions undergoing targeted neuronal apoptosis, including the TrkB neurotrophin receptor ligands BDNF and NT-4/5. We investigated this hypothesis and found that undifferentiated HiB5 cells lack catalytic trkB neurotrophin receptors at the mRNA and protein levels, while confirming that they express trkC receptors under the same conditions. Taken together, these findings support a progressive sequence of neural precursor differentiation and a spectrum of competence by precursors to respond to instructive microenvironmental signals. (ABSTRACT TRUNCATED)     

Human embryonic stem cell-derived neural precursors develop into neurons and integrate into the host brain

Whether and how in-vitro-produced human neural precursors mature and integrate into the brain are crucial to the utility of human embryonic stem (hES) cells in treating neurological disorders. After transplantation into the ventricles of neonatal immune-deficient mice, hES-cell-derived neural precursors stopped expressing the cell division marker Ki67, except in neurogenic areas, and differentiated into neurons and then glia in a temporal course intrinsic to that of human cells regardless of location. The human cells located in the gray matter became neurons in the olfactory bulb and striatum, whereas those in the white matter produced exclusively glia. Importantly, the grafted human cells formed synapses. Thus, the in-vitro-produced human neural precursors follow their intrinsic temporal program to produce neurons and glia and, in response to environmental signals, generate cells appropriate to their target regions and integrate into the brain.     

Avian neural crest cell fate decisions: a diffusible signal mediates induction of neural crest by the ectoderm.

During neurulation, a region of central ectoderm becomes thickened to form the neural plate which then folds upon itself to generate the neural tube, from which all neurons and glia cells of the central nervous system arise. Neural crest cells form at the border of the neural plate, where it abuts the prospective epidermis. The neural crest is a transient population of cells that undergo an epithelial-mesenchymal transition, become highly migratory and subsequently differentiate into most of the peripheral nervous systems as well as numerous other derivatives. The origin of neural crest cells at the epidermal-neural plate border suggests that an interaction between these two tissues may be involved in neural crest formation. By experimentally juxtaposing prospective epidermis with naive neural plate, we previously showed that an inductive interaction between these tissues can generate neural crest cells. Here, we further characterize the nature of this inductive interaction by co-culturing isolated neural plate and prospective epidermis on opposing sides of polycarbonate filters with differing pore sizes. We find that neural crest cells are generated even when epidermis and neural plate are separated by filters that do not allow cell contact. These results suggest that the epidermal inducer is a diffusible, secreted molecule. We discuss the developmental potential of neural crest precursors and lineage decisions that effect their differentiation into numerous derivatives.     

Development of the Schwann cell lineage: from the neural crest to the myelinated nerve

The myelinating and nonmyelinating Schwann cells in peripheral nerves are derived from the neural crest, which is a transient and multipotent embryonic structure that also generates the other main glial subtypes of the peripheral nervous system (PNS). Schwann cell development occurs through a series of transitional embryonic and postnatal phases, which are tightly regulated by a number of signals. During the early embryonic phases, neural crest cells are specified to give rise to Schwann cell precursors, which represent the first transitional stage in the Schwann cell lineage, and these then generate the immature Schwann cells. At birth, the immature Schwann cells differentiate into either the myelinating or nonmyelinating Schwann cells that populate the mature nerve trunks. In this review, we will discuss the biology of the transitional stages in embryonic and early postnatal Schwann cell development, including the phenotypic differences between them and the recently identified signaling pathways, which control their differentiation and maintenance. In addition, the role and importance of the microenvironment in which glial differentiation takes place will be discussed.