Origin of new glial cells in intact and injured adult spinal cord

Several distinct cell types in the adult central nervous system have been suggested to act as stem or progenitor cells generating new cells under physiological or pathological conditions. We have assessed the origin of new cells in the adult mouse spinal cord by genetic fate mapping. Oligodendrocyte progenitors self-renew, give rise to new mature oligodendrocytes, and constitute the dominating proliferating cell population in the intact adult spinal cord. In contrast, astrocytes and ependymal cells, which are restricted to limited self-duplication in the intact spinal cord, generate the largest number of cells after spinal cord injury. Only ependymal cells generate progeny of multiple fates, and neural stem cell activity in the intact and injured adult spinal cord is confined to this cell population. We provide an integrated view of how several distinct cell types contribute in complementary ways to cell maintenance and the reaction to injury.     

Endogenous neurogenesis in adult mammals after spinal cord injury

During the whole life cycle of mammals,new neurons are constantly regenerated in the subgranular zone of the dentate gyrus and in the subventricular zone of the lateral ventricles.Thanks to emerging methodologies,great progress has been made in the characterization of spinal cord endogenous neural stem cells(ependymal cells) and identification of their role in adult spinal cord development.As recently evidenced,both the intrinsic and extrinsic molecular mechanisms of ependymal cells control the sequential steps of the adult spinal cord neurogenesis.This review introduces the concept of adult endogenous neurogenesis,the reaction of ependymal cells after adult spinal cord injury(SCI),the heterogeneity and markers of ependymal cells,the factors that regulate ependymal cells,and the niches that impact the activation or differentiation of ependymal cells.     

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.     

Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury

The adult spinal cord harbours a population of multipotent neural precursor cells (NPCs) with the ability to replace oligodendrocytes. However, despite this capacity, proliferation and endogenous remyelination is severely limited after spinal cord injury (SCI). In the post-traumatic microenvironment following SCI, endogenous spinal NPCs mainly differentiate into astrocytes which could contribute to astrogliosis that exacerbate the outcomes of SCI. These findings emphasize a key role for the post-SCI niche in modulating the behaviour of spinal NPCs after SCI. We recently reported that chondroitin sulphate proteoglycans (CSPGs) in the glial scar restrict the outcomes of NPC transplantation in SCI by reducing the survival, migration and integration of engrafted NPCs within the injured spinal cord. These inhibitory effects were attenuated by administration of chondroitinase (ChABC) prior to NPC transplantation. Here, in a rat model of compressive SCI, we show that perturbing CSPGs by ChABC in combination with sustained infusion of growth factors (EGF, bFGF and PDGF-AA) optimize the activation and oligodendroglial differentiation of spinal NPCs after injury. Four days following SCI, we intrathecally delivered ChABC and/or GFs for seven days. We performed BrdU incorporation to label proliferating cells during the treatment period after SCI. This strategy increased the proliferation of spinal NPCs, reduced the generation of new astrocytes and promoted their differentiation along an oligodendroglial lineage, a prerequisite for remyelination. Furthermore, ChABC and GF treatments enhanced the response of non-neural cells by increasing the generation of new vascular endothelial cells and decreasing the number of proliferating macrophages/microglia after SCI. In conclusions, our data strongly suggest that optimization of the behaviour of endogenous spinal NPCs after SCI is critical not only to promote endogenous oligodendrocyte replacement, but also to reverse the otherwise detrimental effects of their activation into astrocytes which could negatively influence the repair process after SCI.     

Morphology of regenerated spinal cord in Sternarchus albifrons

Summary The tail of the gymnotid Sternarchus albifrons, including the spinal cord, regenerates following amputation. Regenerated spinal cord shows a rostro-caudal gradient of differentiation. Cross sections of the most distal regenerated cord show radially enlarged ependymal cells, relatively undifferentiated cells, and numerous blood vessels. More anterior sections contain well differentiated electromotor neurons, glial cells, and myelinated axons. The number of electromotor-neuron cell bodies in cross sections of regenerated spinal cord is three to six times the number in nonregenerated cord. Distinct tracts of axons, easily identifiable in normal cord, are not distinguishable in cross sections of regenerated cord. Some reorganization of the spinal cord also appears to take place anterior to the site of transection.Individual electromotor neurons in the regenerated spinal cord have morphologies largely similar to those of normal electrocytes, i.e., cell bodies are rounded, lack dendrites, have synapses characterized by gap junctions with presynaptic axons, and lack an unmyelinated initial segment. The presence of electromotor neurons with normal morphology in regenerated spinal cord correlates with the re-establishment of relatively normal electrocyte axonSchwann cell relationships in the regenerating electric organ of this sternarchid.Supported in part by the Medical Research Service, Veterans Administration and by a grant from the National Institutes of Health. We also thank the Paralyzed Veterans of America for their support. We thank Mary E. Smith and Susan Cameron for excellent technical support     

Neuronal differentiation in vitro from precursor cells of regenerating spinal cord of the adult teleost Apteronotus albifrons

This study documents neuronal differentiation in vitro from undifferentiated precursor cells of caudalmost regenerating spinal cord of the teleost Apteronotus albifrons. At 11 days in vitro, cells from the caudalmost tip of the regenerating cord are flat and polygonal in shape, lack neuronal processes and do not stain with antibody against neuron-specific filaments. At 15 days in vitro, some of the caudalmost cells have developed short, neurite-like processes; at 18 days in vitro, some cells react positively with antibody against neuron-specific filaments. At 26 days in vitro, many of the caudalmost cells have long branching neurites and react positively with anti-neurofilament antibody. Addition of insulin-like growth factor-I to the medium accelerates the process of neuronal differentiation from the caudalmost precursor cells in vitro. The source of these precursor cells is ultimately cells of the ependymal layer of adult spinal cord. Further investigation of the factors that control production and differentiation of these cells will be important in defining the developmental potential possible for vertebrate spinal cord cells and may aid in creating an optimal environment for regeneration of axons within mammalian spinal cord.     

Chondroitinase ABC treatment and the phenotype of neural progenitor cells isolated from injured rat spinal cord

The aim of the present study was to investigate whether enzyme chondroitinase ABC (ChABC) treatment influences the phenotype of neural progenitor cells (NPCs) derived from injured rat spinal cord. Adult as well as fetal spinal cords contain a pool of endogenous neural progenitors cells, which play a key role in the neuroregenerative processes following spinal cord injury (SCI) and hold particular promise for therapeutic approaches in CNS injury or neurodegenerative disorders. In our study we used in vitro model to demonstrate the differentiation potential of NPCs isolated from adult rat spinal cord after SCI, treated with ChABC. The intrathecal delivery of ChABC (10 U/ml) was performed at day 1 and 2 after SCI. The present findings indicate that the impact of SCI resulted in a decrease of all NPCs phenotypes and the ChABC treatment, on the contrary, caused an opposite effect.     

Migration and Differentiation of Neural Progenitor Cells after Recurrent Laryngeal Nerve Avulsion in Rats

To investigate migration and differentiation of neural progenitor cells (NPCs) from the ependymal layer to the nucleus ambiguus (NA) after recurrent laryngeal nerve (RLN) avulsion. All of the animals received a CM-DiI injection in the left lateral ventricle. Forty-five adult rats were subjected to a left RLN avulsion injury, and nine rats were used as controls. 5-Bromo-2-deoxyuridine (BrdU) was injected intraperitoneally. Immunohistochemical analyses were performed in the brain stems at different time points after RLN injury. After RLN avulsion, the CM-DiI+ NPCs from the ependymal layer migrated to the lesioned NA. CM-DiI+/GFAP+ astrocytes, CM-DiI+/DCX+ neuroblasts and CM-DiI+/NeuN+ neurons were observed in the migratory stream. However, the ipsilateral NA included only CM-DiI+ astrocytes, not newborn neurons. After RLN avulsion, the NPCs in the ependymal layer of the 4th ventricle or central canal attempt to restore the damaged NA. We first confirm that the migratory stream includes both neurons and glia differentiated from the NPCs. However, only differentiated astrocytes are successfully incorporated into the NA. The presence of both cell types in the migratory process may play a role in repairing RLN injuries.     

Lithium enhances the neuronal differentiation of neural progenitor cells in vitro and after transplantation into the avulsed ventral horn of adult rats through the secretion of brain-derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo . Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.     

Central Canal Ependymal Cells Proliferate Extensively in Response to Traumatic Spinal Cord Injury but Not Demyelinating Lesions

The adult mammalian spinal cord has limited regenerative capacity in settings such as spinal cord injury (SCI) and multiple sclerosis (MS). Recent studies have revealed that ependymal cells lining the central canal possess latent neural stem cell potential, undergoing proliferation and multi-lineage differentiation following experimental SCI. To determine whether reactive ependymal cells are a realistic endogenous cell population to target in order to promote spinal cord repair, we assessed the spatiotemporal dynamics of ependymal cell proliferation for up to 35 days in three models of spinal pathologies: contusion SCI using the Infinite Horizon impactor, focal demyelination by intraspinal injection of lysophosphatidylcholine (LPC), and autoimmune-mediated multi-focal demyelination using the active experimental autoimmune encephalomyelitis (EAE) model of MS. Contusion SCI at the T9–10 thoracic level stimulated a robust, long-lasting and long-distance wave of ependymal proliferation that peaked at 3 days in the lesion segment, 14 days in the rostral segment, and was still detectable at the cervical level, where it peaked at 21 days. This proliferative wave was suppressed distal to the contusion. Unlike SCI, neither chemical- nor autoimmune-mediated demyelination triggered ependymal cell proliferation at any time point, despite the occurrence of demyelination (LPC and EAE), remyelination (LPC) and significant locomotor defects (EAE). Thus, traumatic SCI induces widespread and enduring activation of reactive ependymal cells, identifying them as a robust cell population to target for therapeutic manipulation after contusion; conversely, neither demyelination, remyelination nor autoimmunity appears sufficient to trigger proliferation of quiescent ependymal cells in models of MS-like demyelinating diseases.     

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord

Although the central nervous system is considered a comparatively static tissue with limited cell turnover, cells with stem cell properties have been isolated from most neural tissues. The spinal cord ependymal cells show neural stem cell potential in vitro and in vivo in injured spinal cord. However, very little is known regarding the ependymal niche in the mouse spinal cord. We previously reported that a secreted factor, chick Akhirin, is expressed in the ciliary marginal zone of the eye, where it works as a heterophilic cell‐adhesion molecule. Here, we describe a new crucial function for mouse Akhirin (M‐AKH) in regulating the proliferation and differentiation of progenitors in the mouse spinal cord. During embryonic spinal cord development, M‐AKH is transiently expressed in the central canal ependymal cells, which possess latent neural stem cell properties. Targeted inactivation of the AKH gene in mice causes a reduction in the size of the spinal cord and decreases BrdU incorporation in the spinal cord. Remarkably, the expression patterns of ependymal niche molecules in AKH knockout (AKH?/?) mice are different from those of AKH+/+, both in vitro and in vivo. Furthermore, we provide evidence that AKH expression in the central canal is rapidly upregulated in the injured spinal cord. Taken together, these results indicate that M‐AKH plays a crucial role in mouse spinal cord formation by regulating the ependymal niche in the central canal. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 494–504, 2015     

Nogo-66 promotes the differentiation of neural progenitors into astroglial lineage cells through mTOR-STAT3 pathway

Background

Neural stem/progenitor cells (NPCs) can differentiate into neurons, astrocytes and oligodendrocytes. NPCs are considered valuable for the cell therapy of injuries in the central nervous system (CNS). However, when NPCs are transplanted into the adult mammalian spinal cord, they mostly differentiate into glial lineage. The same results have been observed for endogenous NPCs during spinal cord injury. However, little is known about the mechanism of such fate decision of NPCs.

Methodology/Principal Findings

In the present study, we have found that myelin protein and Nogo-66 promoted the differentiation of NPCs into glial lineage. NgR and mTOR-Stat3 pathway were involved in this process. Releasing NgR from cell membranes or blocking mTOR-STAT3 could rescue the enhanced glial differentiation by Nogo-66.

Conclusions/Significance

These results revealed a novel function of Nogo-66 in the fate decision of NPCs. This discovery could have profound impact on the understanding of CNS development and could improve the therapy of CNS injuries.     

An Engineered N-Cadherin Substrate for Differentiation,Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors

For stem cell-based treatment of neurodegenerative diseases a better understanding of key developmental signaling pathways and robust techniques for producing neurons with highest homogeneity are required. In this study, we demonstrate a method using N-cadherin-based biomimetic substrate to promote the differentiation of mouse embryonic stem cell (ESC)- and induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) without exogenous neuro-inductive signals. We showed that substrate-dependent activation of N-cadherin reduces Rho/ROCK activation and β-catenin expression, leading to the stimulation of neurite outgrowth and conversion into cells expressing neural/glial markers. Besides, plating dissociated cells on N-cadherin substrate can significantly increase the differentiation yield via suppression of dissociation-induced Rho/ROCK-mediated apoptosis. Because undifferentiated ESCs and iPSCs have low affinity to N-cadherin, plating dissociated cells on N-cadherin-coated substrate increase the homogeneity of differentiation by purging ESCs and iPSCs (~30%) from a mixture of undifferentiated cells with NPCs. Using this label-free cell selection approach we enriched differentiated NPCs plated as monolayer without ROCK inhibitor. Therefore, N-cadherin biomimetic substrate provide a powerful tool for basic study of cell—material interaction in a spatially defined and substrate-dependent manner. Collectively, our approach is efficient, robust and cost effective to produce large quantities of differentiated cells with highest homogeneity and applicable to use with other types of cells.     

Explant cultures of teleost spinal cord: source of neurite outgrowth

The source of neurite outgrowth in explant cultures of normal adult Apteronotus spinal cord was examined. Explants which contained the central region of spinal cord, including ependyma, showed neurite outgrowth in culture. Explants which did not contain ependyma showed no neurite outgrowth. It is concluded that the ependymal region is necessary for neurite outgrowth in these cultures of adult teleost spinal cord. In addition, our failure to observe axon outgrowth clearly attributable to fluorescently back-labeled electromotor neurons in these cultures suggests that the exuberant neurite outgrowth in vitro is most probably due to cells other than the electromotor neurons. This explant culture system provides a unique opportunity to study neuronal differentiation, regeneration, and neurogenesis in vitro.     

Mixed primary culture and clonal analysis provide evidence that NG2 proteoglycan-expressing cells after spinal cord injury are glial progenitors

NG2(+) cells in the adult rat spinal cord proliferate after spinal cord injury (SCI) and are postulated to differentiate into mature glia to replace some of those lost to injury. To further study these putative endogenous precursors, tissue at 3 days after SCI or from uninjured adults was dissociated, myelin partially removed and replicate cultures grown in serum-containing or serum-free medium with or without growth factors for up to 7 days in vitro (DIV). Cell yield after SCI was 5-6 times higher than from the normal adult. Most cells were OX42(+) microglia/macrophages but there were also more than twice the normal number of NG2(+) cells. Most of these coexpressed A2B5 or nestin, as would be expected for glial progenitors. Few cells initially expressed mature astrocyte (GFAP) or oligodendrocyte (CC1) markers, but more did at 7 DIV, suggesting differentiation of glial precursors in vitro. To test the hypothesis that NG2(+) cells after SCI express progenitor-like properties, we prepared free-floating sphere and single cell cultures from purified suspension of NG2(+) cells from injured spinal cord. We found that sphere cultures could be passaged in free-floating subcultures, and upon attachment the spheres clonally derived from an acutely purified single cell differentiated into oligodendrocytes and rarely astrocytes. Taken together, these data support the hypothesis that SCI stimulates proliferation of NG2(+) cells that are glial progenitor cells. Better understanding the intrinsic properties of the NG2(+) cells stimulated by SCI may permit future therapeutic manipulations to improve recovery after SCI.