Current status of experimental cell replacement approaches to spinal cord injury

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✓ Despite advances in medical and surgical care, the current clinical therapies for spinal cord injury (SCI) are largely ineffective. During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for a stem cell–based reparative approaches to many diseases, including neurotrauma. In the present study, the authors briefly summarize current knowledge related to the pathophysiology of SCI, including the concepts of primary and secondary injury and the importance of posttraumatic demyelination. Key inhibitory obstacles that impede axonal regeneration include the glial scar and a number of myelin inhibitory molecules including Nogo. Recent advancements in cell replacement therapy as a therapeutic strategy for SCI are summarized. The strategies include the use of pluripotent human stem cells, embryonic stem cells, and a number of adult-derived stem and progenitor cells such as mesenchymal stem cells, Schwann cells, olfactory ensheathing cells, and adult-derived neural precursor cells. Although current strategies to repair the subacutely injured cord appear promising, many obstacles continue to render the treatment of chronic injuries challenging. Nonetheless, the future for stem cell–based reparative strategies for treating SCI appears bright.

Abbreviations used in this paper: CNS = central nervous system; CSPG = chondroitin sulphate proteoglycan; ESC = embryonic stem cell; GFAP = glial fibrillary acidic protein; hESC = human ESC; MBP = myelin basic protein; MSC = mesenchymal stem cell; NPC = neural precursor cell; OEC = olfactory ensheathing cell; PNS = peripheral nervous system; ROS = reactive oxygen species; SCI = spinal cord injury; YFP = yellow fluorescence protein.

✓ Despite advances in medical and surgical care, the current clinical therapies for spinal cord injury (SCI) are largely ineffective. During the last 2 decades, the search for new therapies has been revolutionized by the discovery of stem cells, which has inspired scientists and clinicians to search for a stem cell–based reparative approaches to many diseases, including neurotrauma. In the present study, the authors briefly summarize current knowledge related to the pathophysiology of SCI, including the concepts of primary and secondary injury and the importance of posttraumatic demyelination. Key inhibitory obstacles that impede axonal regeneration include the glial scar and a number of myelin inhibitory molecules including Nogo. Recent advancements in cell replacement therapy as a therapeutic strategy for SCI are summarized. The strategies include the use of pluripotent human stem cells, embryonic stem cells, and a number of adult-derived stem and progenitor cells such as mesenchymal stem cells, Schwann cells, olfactory ensheathing cells, and adult-derived neural precursor cells. Although current strategies to repair the subacutely injured cord appear promising, many obstacles continue to render the treatment of chronic injuries challenging. Nonetheless, the future for stem cell–based reparative strategies for treating SCI appears bright.

Abbreviations used in this paper: CNS = central nervous system; CSPG = chondroitin sulphate proteoglycan; ESC = embryonic stem cell; GFAP = glial fibrillary acidic protein; hESC = human ESC; MBP = myelin basic protein; MSC = mesenchymal stem cell; NPC = neural precursor cell; OEC = olfactory ensheathing cell; PNS = peripheral nervous system; ROS = reactive oxygen species; SCI = spinal cord injury; YFP = yellow fluorescence protein.

According to the Spinal Cord Injury Information Network (in 2006), there are ~ 11,000 new cases of SCI annually, and more than 253,000 people are living with devastating disabilities resulting from SCI. Currently, the average age of a patient at the time of injury is 38.0 years. Thus, SCI affects individuals in the prime of their lives, causing detrimental effects on their quality of life, and a considerable financial burden on society. In light of recent advances in regenerative medicine, the use of stem cells to treat injury and disease has become potentially feasible. However, the availability of suitable stem cells and the complexity of neurological disorders continue to pose significant challenges for cell-replacement therapy applications. Immediate potential targets for stem cell therapy in the CNS include various neurodegenerative diseases, such as Parkinson disease, amyotrophic lateral sclerosis, traumatic brain injuries, and SCIs in which specific cell types are needed to replace the damaged cells.

In this review, we first describe the pathophysiology of the SCI to achieve a better understanding of the mechanisms of SCI and the basis for current cell-based treatment approaches. We then summarize the recent cell therapeutic strategies and findings, including those from our own laboratory, on the use of adult neural stem and progenitor cells for the repair of traumatic SCI and dysmyelinating disorders.

Pathophysiology of SCI

Primary and Secondary Injury Mechanisms

Research efforts over the past 2 decades have expanded our knowledge of the complex pathobiology of SCI (Table 1). Spinal cord injury involves an initial mechanical or primary injury followed by a series of cellular and molecular secondary events resulting in progressive destruction of spinal cord tissue. The wave of secondary cell death, which mainly affects neurons and oligodendrocytes, spreads rostrally and caudally from the site of impact, leading to structural and functional disturbance of the spinal cord. Key secondary injury mechanisms include disruption of spinal cord vasculature and ischemia, glutamatergic excitotoxicity, oxidative cell stress, lipid peroxidation, and inflammation67,96 —all of which alone or in concert can trigger apoptosis.

TABLE 1

Key cellular and molecular events after SCI and potential regenerative approaches*

Key Cellular & Molecular Events After SCI (~ time frames)Underlying Pathobiological MechanismRepair Strategies
cell death type
 necrosis (minutes–hours)oxidative stressantioxidant therapy
 apoptosis (hours–days/weeks)limited availability of endogenous progenitor cells to replace the damaged glia & neuronsmodulation of cell death signaling
modulation of inflammation
cell replacement
neurotrophin administration
modulation of intracellular signaling: Rho pathway
rehabilitation
demyelination (days–months)oligodendrocyte losscell replacement
loss/block of functionneurotrophin administration
glial scar formation (days–weeks)reactive gliosis: ECM remodelingdegradation of ECM molecules
modulation of inflammation
axon degeneration/retraction (days–months)inhibitory/repulsive environment: Nogo, OMG, MAG, CSPGmodulation of intracellular signaling: Rho pathway
loss/block of function
*ECM = extracellular matrix; MAG = myelin-assisted glycoprotein; OMG = oligodendrocyte myelin glycoprotein.

Neuronal and Glial Response to Injury

Neurons and oligodendrocytes are exquisitely vulnerable to secondary cell death after SCI.23 Neurons have a high rate of oxidative metabolism which makes them susceptible to injury by ROS following ischemia.50 Compared to their supporting astroglial cells, neurons have lower levels of antioxidant levels such as glutathione, and respond differently to molecular mechanisms involving the activation of Phase II enzymes responsible for neutralization of damaging free radicals.33 Oligodendrocytes are also very susceptible to ROS, due to their higher iron content and lower levels of glutathione and its related antioxidant enzymes.50 The ROS then initiates a cascade of oxidative events that lead to cell death due to a combination of necrosis and apoptosis, causing degeneration of the gray matter and disruption of the local spinal cord circuits within the injury center.24 During the early days to weeks after injury to the damaged neurons, severed axons and extracellular elements of the necrotic core at the site of injury will be removed by recruited inflammatory cells and phagocytes,32 resulting in the formation of fluid-filled cystic cavities at the site of injury. In approximately 30% of patients, these areas of myelomalacia expand and result in posttraumatic syringomyelia.41

Gliosis results from activation of glial cells, mainly astrocytes, in response to injury. The subsequent increased expression of GFAP, coupled with enhanced astrocytic cell migration and proliferation, results in glial scarring. The glial scar evolves over time to contain the lesion. Although this glial reaction may be partly beneficial in minimizing the extent of lesion size and assisting in the reestablishment of the blood–brain barrier,17 the phenotypic changes in the astrocytic population are mostly inhibitory to the regeneration of severed axons.86 The inhibitory nature of glial scar components will be discussed later in this review in greater detail.

Posttraumatic Changes in Spinal Cord White Matter

Axonal Degeneration

Posttraumatic degradation of white matter results in the loss of volitional motor and sensory control and autonomic dysfunction coupled with aberrant sprouting that can lead to spasticity and neuropathic pain.16, 62,73,76 Axonal degeneration after SCI includes local disruption of the axonal membrane, swelling of axonal mito-chondria, formation of “nodal blebs,” loss of microtubules, compaction and loss of neurofilament side arms, and axonal disconnection.16,62,76 The part of the axon distal to the injury site is degenerated by phagocytosis, but the proximal segment survives. Spontaneous regeneration of the proximal part is a natural phenomenon in the PNS; however, CNS axons lack this property, and only limited sprouting in the spared axons may occur that can contribute to some functional recovery.106

Demyelination

A complete transection of the spinal cord is rare. Even after severe SCI, the spared white matter at the site of injury often includes a subpial rim of surviving small-diameter axons with varying degrees of demyelination as a result of excitotoxicity-mediated cell death of the oligodendrocytes.75 Subsequently the failure of endogenous oligodendrocytes to effect significant remyelination leads to a relative degree of demyelination of spinal cord axons in the chronic stages of SCI.100

Myelin facilitates fast “saltatory conduction” of action potentials and plays a key role in determining the molecular organization of axons, as has been reviewed previously.67 Myelinated fibers display a highly organized molecular architecture in which the Na+ channels are contained at the nodal area, and Kv1.1 and Kv1.2 K+ channel subunits are located under the compact myelin in the juxtaparanodal zones (Fig. 1). The nodal and juxtaparanodal regions are separated by the paranode, which is identified by the presence of the contactin-associated protein. Myelinating glial cells form septate like junctions with the paranodal axonal membrane; these appear to be the key to precise localization of ion channels on axons.84

Fig. 1.
Fig. 1.

Diagram demonstrating the molecular architecture of the myelinated and demyelinated spinal cord axons Myelinated fibers display a highly organized molecular architecture, in which the Na+ channels are localized to the nodes of Ranvier and the Kv1.1 and Kv1.2 K+ channel subunits are located under the compact myelin sheets in the juxtaparanodal zones. The nodal and juxtaparanodal regions are separated by the paranode, which is identified by the presence of contactin-associated protein. Myelin loss after injury or dysmyelination results in marked alterations in the molecular organization of axons. Caspr = contactin-associated protein.

Our group and others have shown that the dysfunctional properties of the surviving demyelinated axons are associated with changes in the distribution and expression of Kv1.1 and Kv1.2 K+ channels.4,34,35,52,67,68,92,101 Indeed, axonal conduction deficits in various in vitro and in vivo models of traumatic demyelination or genetic dysmyelination are partially restored after application of 4-aminopyridine, a K+-channel antagonist.2,34,92 However, clinical use of 4-aminopyridine in patients with SCI has significant limitations,5 which gives impetus to the use of cell-based remyelination strategies as a more clinically promising approach.

Inhibitory Obstacles to Axonal Regeneration

Spontaneous regeneration and regrowth of the proximal segment of the injured spinal cord has been an important target for translational research.99 Several key players such as glial cells and their products contribute to the inhibitory environment of the spinal cord. Models of complete spinal cord transection have played an important role in identifying these underlying mechanisms, although such models may not represent the actual events after traumatic SCI. Some of the key mechanisms that hinder the current approaches for regeneration studies are discussed here.

Glial Scar

Formation of the glial scar represents an attempt by glial cells to contain the injury site and promote healing. Increasing evidence points to a number of molecular similarities between the SCI and dermal wound healing processes.104 In addition to reactive astrocytes, scar formation also involves oligodendrocyte precursor cells, microglia, and macrophages.21 After injury, upregulation of CSPGs, which are associated with astrocytes and oligodendrocyte precursors, is a major contributor to the inhibitory properties of the adult CNS glial scar.109 These molecules comprise large molecular complexes formed by highly sulphated glycosaminoglycans attached to a central protein core. The inhibitory effect of the glial scar has been reduced significantly by enzymatic separation of glycosaminoglycan side chains from the protein core.12 The formation of CSPGs can also be inhibited by molecular degradation of the related enzymes,42 thereby allowing axonal growth in injured spinal cord. Although no receptor has been identified for CSPGs, it has been shown that various signaling pathways are involved in CSPG-mediated inhibition including the Rho–Rock pathway63 and activation of epidermal growth factor receptors.56

Myelin/Oligodendrocyte Inhibitory Molecules

The inhibitory effect of myelin/oligodendrocyte inhibitory molecules has been studied extensively.89,90 The Nogo family (Nogo-A, B, and C) of oligodendrocyte membrane proteins provides some of the best-studied inhibitory signals for neurite outgrowth. The inhibitory function of Nogo proteins lies in 2 structural domains: the amino terminal region, unique to Nogo-A; and a 66-base amino acid loop which is commonly expressed in all 3 isoforms.109 Nogo-A is expressed in oligodendrocytes, and its neutralization by a monoclonal antibody in the acutely injured CNS results in axonal regeneration and functional improvement.13,20 The regrowth of the severed axons and apparent functional synapses has been shown in these models, and was associated with an improved functional outcome.

Myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, semaphorin 4D, and ephrin B3 are other identified inhibitory molecules in the oligodendrocyte membrane. Myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and Nogo-A use a common signaling mechanism to apply their inhibitory effects on axonal regrowth. A battery of neuronal membrane proteins including Nogo receptor, p75, TROY, and LINGO59 has been shown to relay the inhibitory message to the neuronal internal signaling system, through a set of small guanosine triphosphatases such as RhoA, Rac1, and Cdc42. Inhibition of RhoA using a dominant negative approach seems to be the most promising tool to overcome the inhibitory nature of the CNS myelin. The addition of a Rho-selective antagonist, C3-05 transferase, injected into a fibrin matrix to the lesion site in a transection cord injury model, or C3-05 alone injected into the contused cord effectively reduced the RhoA activation levels back to physiological conditions.31 This was associated with reduced neuronal and glial cell apoptosis.31 A Phase I/IIa multinational clinical trial has been recently completed using Cethrin (BioAxone Therapeutic, Inc.), a derivative of C3.5 The preliminary results of this trial are very promising, and the final results are expected to be announced in 2008.

Recent Advances in Cell Replacement Therapy for SCI

Cell death is inevitable after SCI. The adult spinal cord harbors endogenous stem/progenitor cells, collectively referred to as NPCs, that might be responsible for normal turnover of the cells. However, the proliferative activity of endogenous NPCs is too limited to support significant self-repair after SCI. Thus, various cellular transplantation strategies have been adopted in models of SCI.

Cell replacement approaches in the setting of SCI can be used to achieve 2 broad goals: 1) regeneration, which seeks to replace lost or damaged neurons and induce axonal regeneration or plasticity; and 2) repair, which seeks to replace supportive cells such as oligodendrocytes in order to induce remyelination and prevent progressive myelin loss.100 Alternatively, cell transplantation may promote protection of endogenous cells from further secondary injury.

Although the presence of developmental inhibitory or repulsive cues such as Netrin1,78 semaphorin 4D, and ephrin B3 in the adult CNS may complicate the successful restoration of various neuronal components of the spinal cord at the injury site, the specificity of oligodendrocytic cell death in white matter pathologies has attracted attention as a translationally relevant target for experimental SCI treatments. In the following sections we review the current knowledge on cell replacement therapies after SCI. A summary of cell therapy approaches has been listed in Table 2.

TABLE 2

Summary of key cellular approaches to repair and regeneration after SCI*

Cell SourceTransplanted Cell TypeReported Outcome
ESC/progenitor cellsESCs (pluripotent)generation of endothelial cells, neurons, astrocytes, & oligodendrocytes103
embryonic-derived progenitor cells (unipotent)oligodendrocyte & enhanced myelination54
fetal stem/progenitor cells (multipotential)fetal stem/progenitor NPCsastrocytes8
fetal OECscontroversial outcome9
umbilical cord blood stem/progenitor cellsneurons & oligodendrocytes generated, & decreased apoptosis26
no survival 3 weeks posttransplant70
adult cells (pluripotent, multipotent, & unipotent)mesenchymal stem/progenitor cells bone marrow stem cellsneurons, astrocytes, oligodendrocytes25
adipose tissuesneurons, oligodendrocytes & enhanced myelination51
adult NPCsneurons, oligodendrocytes, astrocytes, & enhanced myelination53
Schwann/OECs/SKPsSchwann cell myelination7,11,87
*SKP = skin-derived precursor cell.

Human Pluripotent Stem Cells

The derivation of hESCs in 1998 with their pluripotential ability to produce all cell types of the human body98 has sparked significant interest in their use as therapeutic tools. Considering the complexity of the nervous tissue and the importance of nonneuronal elements, the pluripotentiality of hESCs makes them an optimal candidate that can potentially differentiate into different cell types for the reformation of cellular networks.

Using an excitotoxicity brain injury models,103 it has been shown that transplanted hESCs express immunological labeling for vascular endothelial cells, glia, and different neuronal subtypes with morphologically normal synapses. Recent reports on the generation of functionally efficient motor neurons from ESCs in experimental models of motor neuron diseases such as amyotrophic lateral sclerosis show additional promise for applications of ESCs.69

Currently, generation of these cells requires destruction of early human embryos, which has resulted in ethical concerns and legal issues that have overshadowed the potential of these cells as a cure for almost any disease, disorder, or injury. However, the production of hESCs from a single blastomere obtained at the morula stage with no effect on the embryo's developmental potential has been reported recently.55 Despite these advances, there are several other questions associated with hESCs, including the recipient immune response. It has been reported that undifferentiated hESCs, which have low levels of expression of human leukocyte antigen Class I and express no Class II antigens,18 might be tolerated by the host tissue even after some early differentiation.58 However it is not clear whether fully differentiated hESCs might still maintain their immune-privileged property. Properties similar to those of cancer cells such as genetic instability,43 lack of senescence,111 and increased DNA repair activity might be attributable to increased proliferative capacity and tumorigenesis, causing major concerns that limit the use of undifferentiated hESCs in experimental transplantation.83 Tumor formation might be obviated through microencapsulation of the ESCs.27 This could be useful for diseases such as diabetes, in which direct contact of the transplanted cells with the host tissue is not required. However, application of such approaches for nervous system repair seems unlikely. Using vigorous in vitro cell selection and molecular approaches, mature glial cells can be produced from the ESCs47,112 and have been shown to successfully and effectively myelinate15 spinal cord axons after injury;71 however, the potential for tumorigenesis is always a concern in the application of cell therapy in patients.

Recently the generation of autologous pluripotent stem cells from skin cells has been reported.110 By using viral gene transfer into somatic cells of adult humans, pluripotent cells can be generated that have similar immunological characteristics to the donor. These pluripotent cells are similar to ESCs in that they retain the developmental potential to differentiate into any cell type. This discovery will obviate the ethical issues surrounding the source of ESCs and eliminate the need for immunological matching between the host and donor. However, several issues including the potential mutation of these cells after integration of the carrier viral gene into the host DNA must be resolved before these cells can be used in clinical trials.

Fetal Stem Cells

Fetal tissue grafts were tested in human patients with CNS disorders and Parkinson disease as early as the 1970s.30 Some recent reports have suggested that fetal-derived NPCs can differentiate into neurons and oligodendrocytes after transplantation into a contusion SCI model with some improved behavioral outcome.72,95 The transplantation of OECs from fetal sources, popularized by Huang and colleagues28 in China, has gained considerable media attention; however, the limited scientific data from this study severely limit enthusiasm for this procedure as a therapeutic option in patients. In view of limited availability of fetal sources and the ethical issues surrounding their use, large-scale use of this source as a treatment option remains challenging.

In recent years, banking of umbilical cord blood cells, another type of fetal stem cells, for the cure of immunological and hematological disorders as well as a potential source for stem-cell based therapeutic approaches, has expanded rapidly. The multipotentiality of these cells to produce the main cell types of nervous tissue has been recently reported.25,57 These cells have also been used in experimental SCI models and were shown to improve functional recovery due to neurotrophic function70 or increased myelination.25 However, further work must be done to fully characterize the extent of functional recovery induced by umbilical cord blood cells and to further delineate their mechanisms of action.

Adult-Derived Stem and Progenitor Cells

Adult-derived stem/progenitor cells have been identified in many tissues including those from skin, eye, pancreas, brain, and spinal cord. These somatic cells have an extensive capacity for self-renewal and the multipotentiality to produce all the major cell types of their specialized tissue of origin. However, experimental evidence has suggested the possibility of transdifferentiation of these cells into other cell types, when transplanted ectopically into other tissues such as the spinal cord. Although this topic is the subject of active research, in this review we focus on selected reports that have shown some beneficial outcome when adult cells were transplanted into an SCI model. The major advantage of adult stem and progenitor cells is that their use avoids the potential ethical and biological issues surrounding the use of ESCs and fetal-derived stem cells for regenerative therapies.

Schwann Cells and OECs

The PNS myelinating Schwann cells would seem to be an obvious substitute when considering a cell-based remyelination strategy. Interestingly, infiltration of endogenous Schwann cells at the site of injury and their contribution to spontaneous myelination after SCI is well documented,94 although the impact of such schwannosis—which is associated with increased CSPG formation14 and may have deleterious effects on regeneration—is unclear and could even promote neuropathic pain. Schwann cells can be easily harvested from the patient's peripheral nerves, and a relatively short time is required to produce clinically suitable material for autologous transplantation.

After PNS injury, Schwann cells provide a growth-promoting environment for regenerating axons through secretion of neurotrophic factors1 and extracellular matrix elements22 and ultimately myelinate the axons. Several groups have studied transplantation of Schwann cells after SCI and have shown successful integration of these cells in the host tissue with some regeneration of sensory axons and myelination of these axons associated with modest improvement of hindlimb function.74 A poor survival rate has been reported for these cells when transplanted into the epicenter cavity, although the endogenous Schwann cells will fill up the cavity.6,77 Schwann cells derived from the skin have also been used in an experimental model of SCI.7 Showing a modest functional recovery, the authors showed that the skin-derived precursor cells survive in the cavity and may induce regeneration and myelination of some axons; however, the functionality of these axons and their contribution to the improved function has not been addressed. The therapeutic effects of Schwann cells have been shown to be enhanced when these cells are transplanted with their phenotypically similar cells, OECs. These are peripheral glial cells that support the growth of newly generated axons from the olfactory neurons to the olfactory bulb but do not myelinate the olfactory axons in their native environment.29 However, some authors believe that OECs are not only involved in axonal regeneration but also in remyelination after SCI or in a demyelinating disease.88 In contrast, others have demonstrated a lack of myelination in axons associated with OECs transplanted after SCI.9,77 The controversial myelinating ability of OECs is affected by several experimental variables including the age of the donor9 and the isolation methods used. The OECs and Schwann cells express p75, which is commonly used for OEC purification. Therefore, the contamination of OECs with Schwann cells may partly account for the contradicting observations concerning the ability of OECs to remyelinate axons. Using proteomic techniques, calponin has been found to be specifically expressed by OECs,48 and the results of electron microscopy have suggested that OECs are mainly supportive cells for axonal regeneration, providing a structural framework for the endogenous Schwann cells to promote myelination.10,11

Mesenchymal Stem Cells

These cells are derived from the embryonic mesodermal layer and retain the cardinal abilities of stem cells for self-renewal and multipotentiality to differentiate to the various connective tissue cell types. Mesenchymal stem cells can be obtained from bone marrow, adipose tissue, peripheral and umbilical cord blood, and muscle tissue. These sources thus provide a readily available autologous basis for cell transplantation, alleviating the need for long-term immunosuppression. Mesenchymal stem cells have been used in experimental models of SCI and in preliminary clinical trials for SCI44,93 with apparent improvement of behavioral outcome. The ability of these cells to differentiate into neural cells is controversial; therefore, it has been suggested that the functional improvement might be effected through neuroprotective pathways, perhaps by inhibition of Fas-mediated apoptosis.26 As reviewed recently,80 several groups have shown the in vivo expression of neural tissue markers nestin and GFAP in MSCs or their neuron like morphological characteristics under certain tissue culture conditions as evidence of the transdifferentiation ability of MSCs to neural tissue cells. Nonetheless, nestin and GFAP are not specific markers for nervous tissue and can be found in mesodermal tissues such as muscle and cartilage. The reported neuron-like morphological characteristics of MSCs after tissue culture manipulation can best be described as tissue-culture artifact and has not been supported by functional properties associated with normal neurons such as electrophysiological assessment. The possibility of MSC transdifferentiation into neuronal progeny remains to be investigated.80 Alternatively the functional benefits of MSC transplantation in CNS injuries can be explained by their ability to provide the host tissue with growth factors or modulate the host immune system.37 These cells can be used in combinatorial therapies as tools for growth factor delivery. In fact, Lu and colleagues60 have reported that transplantation of bone marrow MSCs, engineered to deliver neurotrophins, can induce axonal growth through the glial scar in a model of chronic SCI in rats; however, it seems that these approaches do not result in functional recovery after chronic SCI, indicating the lack of appropriate synapse formation by the regenerated axons with the host tissue.

Adult-Derived NPCs

Adult NPCs contribute to neurogenesis and gliogenesis in some regions of the CNS in various species including humans.38,39 These tissue-specific somatic stem cells are located in the periventricular regions3,46,65,85,97,107 and in white matter tissue38,40,81,82 throughout the brain and spinal cord and can differentiate into all neural cell types under defined conditions.49,91,107,108 Using selective in vitro expansion and cell sorting based on antigenic properties and molecular techniques, these cells can be directly derived from the adult tissue; however, so far no specific marker has been identified for human adult NPCs.69

Recently, using a combined therapeutic strategy, we have shown the beneficial effects of adult NPC transplantation in a clinically relevant model of SCI in rats.53 This compressive model consists of a modified aneurysm clip that delivers a calibrated closing force. The 23-g clip used in our studies produces a moderately severe SCI with central cavitation and loss of 80% of axons in the spinal cord white matter,36 demyelination of the surviving axons in the residual subpial rim, and spastic paraparesis.66,67 Therefore, this model is of translational relevance to the majority of patients with chronic SCI.

The adult NPCs were harvested from the subventricular zone of adult mice that express the YFP gene, allowing for easy tracking of transplanted cells. Cells were clonally driven in vitro in the presence of growth factors. These free-floating colonies (called neurospheres) were composed of 1% neural stem cells and 99% progenitor cells.64 We transplanted the dissociated adult NPCs into the areas rostral and caudal to the injury epicenter in a delayed fashion at 2 or 8 weeks postinjury to represent 2 clinically relevant phases in SCI pathophysiology, the subacute and chronic stages, respectively. We adopted a novel posttransplantation delivery of epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor-AA approach aimed at: 1) promoting the survival of the grafted adult NPCs; and 2) in vivo differentiation of undifferentiated NPCs towards an oligodendroglial lineage.

Transplantation of Adult NPCs into the Subacutely Injured Spinal Cord

When transplantation was delayed to 2 weeks after injury, a substantial survival rate of 40% of the transplanted cells was observed (Fig. 2). Our study is one of the few that have reported such a high survival rate, suggesting the efficiency of our growth factor delivery regimen in maximizing cell survival. The multipotent capability of the sub-ventricularly derived adult NPCs to differentiate into the 3 main cell types of the nervous tissue has been shown in our experiments as well as in previous reports in vitro,65,85 but the transplanted NPCs were mainly driven towards a glial fate. The lack of neuronal differentiation by engrafted NPCs in the adult spinal cord has been reported repeatedly by different groups of authors.19,79,105 The tendency of NPCs toward glial differentiation after transplantation may also reflect the intrinsic properties of the spinal cord that favor glial differentiation.61 This may be attributed to potential inhibitory or lack of promoting factors for neurogenesis by NPCs in different CNS tissues and needs to be taken into consideration when cell-therapy strategies are designed for neuronal replacement.

Fig. 2.
Fig. 2.

Photomicrographs demonstrating that transplanted adult NPCs successfully survive, migrate, and integrate with the injured spinal cord. Confocal images from a longitudinal section of injured spinal cord taken from the dorsal spinal cord of an injured rat that received cell transplants 2 weeks postinjury. The low-magnification image (A) shows a substantial survival of YFP-NPCs within the injured spinal cord at 8 weeks posttransplantation. Grafted YFP-NPCs (green) were dispersed along the rostrocaudal axis of the spinal cord approximately 5 mm from the implantation sites (asterisks). The YFP-NPCs also migrated to the contralateral side of the spinal cord to a lesser extent. Double labeling with the neuronal marker βIII tubulin (Tuj1) showed that YFP-NPCs reside predominantly in the white matter (WM) area. GM = gray matter. (Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG: Delayed transplantation of adult neural stem cells promotes remyelination and functional recovery after spinal cord injury. J Neurosci 26:3377–3389, 2006. Copyright 2006 by the Society for Neuroscience.)

We found a clear preference for grafted adult NPCs to remain confined to the white matter (Fig. 2), differentiate along oligodendroglial lineage, ensheath the axons, and form compact myelin (Fig. 3). We have made similar observations when these cells were transplanted into the CNS of dysmyelinated shiverer mice35—genetically dysmyelinated mice lacking MBP and therefore lacking central myelin (Fig. 4). Because loss of oligodendrocytes and myelin is a major contributor to white matter damage after trauma, it is rather intriguing to speculate that the propensity of adult NPCs to form oligodendrocytes after transplantation into the adult injured spinal cord may reflect the influence of the host tissue to replenish a particular cell type to maximize repair. However, whether demyelinated axons exert such effects on NPC integration and differentiation remains to be investigated.

Fig. 3.
Fig. 3.

Transplanted adult NPCs enhance remyelination of the injured spinal cord. Confocal images of longitudinal sections of injured spinal cord 8 weeks after NPC transplantation (A–G) and graph (H). A–C: A demyelinated area in injured spinal cord grafted with YFP-NPCs (green) displays a robust expression of MBP (red) in white matter. Cell bodies of donor cells are surrounded with MBP. D and E: Images taken by deconvolution confocal microscopy confirm the axonal ensheathment of MBP-expressing YFP-NPCs around the injured axons. F and G: The NPC-transplanted group showed more extensive oligodendrocyte-myelinated profiles in the area that was occupied by YFP-NPCs. The presence of YFP-NPCs was confirmed by fluorescence microscopy in the adjacent sections. H: Myelin index measurements showed a significant increase in myelin ratio in the NPC-transplanted group compared with nontransplanted control injured animals, indicating enhanced remyelination by NPC-derived oligodendrocytes. Collectively, these findings suggest the remyelinating ability of adult NPCs for the repair of SCI (Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG: Delayed transplantation of adult neural stem cells promotes remyelination and functional recovery after spinal cord injury. J Neurosci 26:3377–3389, 2006. Copyright 2006 by the Society for Neuroscience).

Fig. 4.
Fig. 4.

Myelination of the dysmyelinated spinal cord axons of shiverer mice by NPC transplantation improves axonal conduction. After NPC transplantation, robust expression of MBP was observed in shiverer spinal cords in the area occupied by NPCs (A–C) as shown on MBP staining. The transplanted adult NPCs differentiate to typical oligodendrocytes, ensheath and myelinate several axons in their vicinity (D), and form nodes of Ranvier (E). Electron microscopic images confirm lack of compact myelin in nontransplanted specimen (F). At 6–8 weeks after transplantation many myelinated axons were observed in transplanted segments of spinal cord axons (G) containing multilayered compact myelin (H and I). In vivo electrophysiological assessment of transplanted shiverer (shi) spinal cord revealed improved conductive properties in transplanted spinal cord segments (J–L) (Eftekharpour E, Karimi-Abdolrezaee S, Wang J, El Beheiry H, Morshead C, Fehlings MG: Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27:3416–3428, 2007. Copyright 2007 by the Society for Neuroscience).

Myelin is critical to the precise molecular organization of axons. The nodal distribution of Na+ channels, paranodal preference of contactin-associated protein, and juxtapara-nodal adherence of K+ channels is closely related to myelin and is disrupted following demyelination. To examine the remyelinating potential of adult NPCs, we used shiverer mice because their lack of myelin makes them an excellent model to study the biological effect of myelin on axonal molecular architecture. With adult NPC transplantation in this model, we showed a significant improvement in the molecular organization of axons and in particular in the restoration of the nodes of Ranvier (Fig. 5). Similar observations have been reported in experimentally induced demyelination in which glial cells are temporarily killed using cytotoxic agents and x-ray irradiation.87 However, the potential contribution of endogenous myelinating cells in these models cannot be ruled out unless mutant dysmyelinated models such as neonatal myelindeficient rats102 or shiverer mice35 are used. Beneficial structural and functional effects of neural stem cells in genetic mutant models indicate that long-term molecular abnormalities can be successfully reversed in the adult dysmyelinated CNS axons.

Fig. 5.
Fig. 5.

Myelination of the dysmyelinated spinal cord axons of shiverer mice restores the axonal molecular structure. The effect of myelin on axonal molecular architecture was confirmed in shiverer mice. In shiverer mice Kv1.2 immunostaining (red) is abnormally distributed along the axonal internodes (A– C); however, Na+ clusters were observed as nodal aggregates (blue). Six weeks after adult NPC transplantation, spinal cord segments of shiverer mice showed restoration of Kv1.2 subunit clusters (D–F). The YFP-positive processes of transplanted adult NPCs were observed in close association with axons containing restored K+ channel aggregates forming node-like structures (G). (Eftekharpour E, Karimi-Abdolrezaee S, Wang J, El Beheiry H, Morshead C, Fehlings MG: Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27:3416–3428, 2007. Copyright 2007 by the Society for Neuroscience).

Although neuroanatomical assessment of myelination is a common approach determining the success of cell-therapy approaches, direct physiological assessment to investigate the functionality of the new myelin has been overlooked in literature. Using in vivo spinal cord evoked potential recordings, we observed that adult NPC-induced myelination promoted enhanced spinal cord axonal conduction in the shiverer mice, as evidenced by an increased amplitude, reduced latency, and enhanced estimated conduction velocity (Fig. 4).

Although remyelination has been shown to be involved in adult NPC-mediated recovery after SCI, we cannot exclude other potential protective/trophic effects of adult NPCs. It is possible that the transplanted adult NPCs may increase axonal sprouting and plasticity that could be responsible for some of the observed locomotor recovery. Functional locomotor recovery following subacute transplantation of adult NPCs has been also reported by other investigators.45 However, recent reports have noted an increase in pain sensation, known as allodynia, which was observed when NPCs chiefly differentiated into astrocytes.45 Allodynia was diminished after genetic modification of the naive adult NPCs to enhance their differentiation towards oligodendrocyte lineage. Of note, we have not observed any changes in pain sensation in our experiments (unpublished data), which can be explained by low rates of astrocytic differentiation by the transplanted adult NPCs in our transplantation protocol. However, these reports suggest that the therapeutic evaluation of any intervention for the repair of SCI should include detailed sensory and motor assessments.

Transplantation of NPCs Into Chronic Spinal Cord Lesions

Chronic SCI continues to pose a major challenge for patients, physicians and translational scientists. To date, limited success has been achieved in applying regenerative technologies to the complex pathobiology of chronic SCI. This may be attributed to the environmental restrictions present in chronically injured spinal cord including glial scarring and syrinx formation. In our experience with adult NPC therapy for the repair of SCI, we have learned that the chronically injured spinal cord environment is not as hospitable as the subacutely injured tissue for the transplanted cells. Grafted adult NPCs mostly failed to survive after transplantation into the chronically injured spinal cord. These results suggest the presence of inhibitory components or the lack of promoters such as growth factors in the environment of chronically injured spinal cord. Considering the complexity of chronic SCI pathobiology, it is important to adopt a multifactorial approach to overcome the environmental restrictions present in the chronically injured spinal cord. Such strategies will need to overcome the inhibitory effects for the glial scar, endogenous inhibitory molecules such as Nogo and Rho, and the presence of posttraumatic cysts.

Conclusions

Current cell-based approaches are aimed at: 1) regeneration of new neurons that die within the first minutes to days after trauma; 2) providing a source of cells to promote remyelination; and 3) delivering trophic molecules that can promote cellular protection and plasticity. Activation of endogenous adult NPCs in response to injury appears to occur to only a very limited extent after SCI and is not sufficient to replace the damaged or lost cells. Many sources of precursor cells have been applied in the setting of SCI, but arguably the most convincing results in preclinical models have been obtained with NPCs. Although the use of cell-based transplantation strategies for the repair of chronic SCI remains the long sought after holy grail, these approaches have been to date most successful when applied in the subacute phase of injury. The application of cell-based strategies for repair and regeneration of the chronically injured spinal cord will require a combinatorial strategy that will probably need to include approaches to overcome the effects of the glial scar, inhibitory molecules such as Rho and Nogo, and the use of tissue engineering strategies to bridge the lesion. Nonetheless, cell transplantation strategies are promising, and it is anticipated that the Phase I clinical trials of some form of neural stem cell–based approach in SCI will commence in the next several years.

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Article Information

Contributor Notes

Address correspondence to: Michael G. Fehlings, M.D., Ph.D., Division of Neurosurgery, University of Toronto, Toronto Western Hospital, University Health Network Room 4W-449, 399 Bathurst Street Toronto, Ontario M5T 2S8. email: michael.fehlings@uhn.on.ca.

© AANS, except where prohibited by US copyright law.

Headings
Figures
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    Diagram demonstrating the molecular architecture of the myelinated and demyelinated spinal cord axons Myelinated fibers display a highly organized molecular architecture, in which the Na+ channels are localized to the nodes of Ranvier and the Kv1.1 and Kv1.2 K+ channel subunits are located under the compact myelin sheets in the juxtaparanodal zones. The nodal and juxtaparanodal regions are separated by the paranode, which is identified by the presence of contactin-associated protein. Myelin loss after injury or dysmyelination results in marked alterations in the molecular organization of axons. Caspr = contactin-associated protein.

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    Photomicrographs demonstrating that transplanted adult NPCs successfully survive, migrate, and integrate with the injured spinal cord. Confocal images from a longitudinal section of injured spinal cord taken from the dorsal spinal cord of an injured rat that received cell transplants 2 weeks postinjury. The low-magnification image (A) shows a substantial survival of YFP-NPCs within the injured spinal cord at 8 weeks posttransplantation. Grafted YFP-NPCs (green) were dispersed along the rostrocaudal axis of the spinal cord approximately 5 mm from the implantation sites (asterisks). The YFP-NPCs also migrated to the contralateral side of the spinal cord to a lesser extent. Double labeling with the neuronal marker βIII tubulin (Tuj1) showed that YFP-NPCs reside predominantly in the white matter (WM) area. GM = gray matter. (Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG: Delayed transplantation of adult neural stem cells promotes remyelination and functional recovery after spinal cord injury. J Neurosci 26:3377–3389, 2006. Copyright 2006 by the Society for Neuroscience.)

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    Transplanted adult NPCs enhance remyelination of the injured spinal cord. Confocal images of longitudinal sections of injured spinal cord 8 weeks after NPC transplantation (A–G) and graph (H). A–C: A demyelinated area in injured spinal cord grafted with YFP-NPCs (green) displays a robust expression of MBP (red) in white matter. Cell bodies of donor cells are surrounded with MBP. D and E: Images taken by deconvolution confocal microscopy confirm the axonal ensheathment of MBP-expressing YFP-NPCs around the injured axons. F and G: The NPC-transplanted group showed more extensive oligodendrocyte-myelinated profiles in the area that was occupied by YFP-NPCs. The presence of YFP-NPCs was confirmed by fluorescence microscopy in the adjacent sections. H: Myelin index measurements showed a significant increase in myelin ratio in the NPC-transplanted group compared with nontransplanted control injured animals, indicating enhanced remyelination by NPC-derived oligodendrocytes. Collectively, these findings suggest the remyelinating ability of adult NPCs for the repair of SCI (Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG: Delayed transplantation of adult neural stem cells promotes remyelination and functional recovery after spinal cord injury. J Neurosci 26:3377–3389, 2006. Copyright 2006 by the Society for Neuroscience).

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    Myelination of the dysmyelinated spinal cord axons of shiverer mice by NPC transplantation improves axonal conduction. After NPC transplantation, robust expression of MBP was observed in shiverer spinal cords in the area occupied by NPCs (A–C) as shown on MBP staining. The transplanted adult NPCs differentiate to typical oligodendrocytes, ensheath and myelinate several axons in their vicinity (D), and form nodes of Ranvier (E). Electron microscopic images confirm lack of compact myelin in nontransplanted specimen (F). At 6–8 weeks after transplantation many myelinated axons were observed in transplanted segments of spinal cord axons (G) containing multilayered compact myelin (H and I). In vivo electrophysiological assessment of transplanted shiverer (shi) spinal cord revealed improved conductive properties in transplanted spinal cord segments (J–L) (Eftekharpour E, Karimi-Abdolrezaee S, Wang J, El Beheiry H, Morshead C, Fehlings MG: Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27:3416–3428, 2007. Copyright 2007 by the Society for Neuroscience).

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    Myelination of the dysmyelinated spinal cord axons of shiverer mice restores the axonal molecular structure. The effect of myelin on axonal molecular architecture was confirmed in shiverer mice. In shiverer mice Kv1.2 immunostaining (red) is abnormally distributed along the axonal internodes (A– C); however, Na+ clusters were observed as nodal aggregates (blue). Six weeks after adult NPC transplantation, spinal cord segments of shiverer mice showed restoration of Kv1.2 subunit clusters (D–F). The YFP-positive processes of transplanted adult NPCs were observed in close association with axons containing restored K+ channel aggregates forming node-like structures (G). (Eftekharpour E, Karimi-Abdolrezaee S, Wang J, El Beheiry H, Morshead C, Fehlings MG: Myelination of congenitally dysmyelinated spinal cord axons by adult neural precursor cells results in formation of nodes of Ranvier and improved axonal conduction. J Neurosci 27:3416–3428, 2007. Copyright 2007 by the Society for Neuroscience).

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