Using Neural Stem Cells to Enhance Repair and Recovery of Spinal Circuits After Injury
Summary and Keywords
Spinal cord injury is characterized by a complex set of events, which include the disruption of connectivity between the brain and the periphery with little or no spontaneous regeneration, resulting in motor, sensory and autonomic deficits. Transplantation of neural stem cells has the potential to provide the cellular components for repair of spinal cord injury (SCI), including oligodendrocytes, astrocytes, and neurons. The ability to generate graft-derived neurons can be used to restore connectivity by formation of functional relays. The critical requirements for building a relay are to achieve long-term survival of graft-derived neurons and promote axon growth into and out of the transplant. Recent studies have demonstrated that mixed populations of glial and neuronal progenitors provide a permissive microenvironment for survival and differentiation of early-stage neurons, but inclusion of growth factors with the transplant or cues for directional axon growth outside the transplant may also be needed. Other important considerations include the timing of the transplantation and the selection of a population of neurons that maximizes the effective transmission of signals. In some experiments, the essential neuronal relay formation has been developed in both sensory and motor systems related to locomotion, respiration, and autonomic functions. Despite impressive advances, the poor regenerative capacity of adult CNS combined with the inhibitory environment of the injury remain a challenge for achieving functional connectivity via supraspinal tracts, but it is possible that recruitment of local propriospinal neurons may facilitate the formation of relays. Furthermore, it is clear that the new connections will not be identical to the original innervation, and therefore there needs to be a mechanism for translating the resulting connectivity into useful function. A promising strategy is to mimic the process of neural development by exploiting the remarkable plasticity associated with activity and exercise to prune and strengthen synaptic connections. In the meantime, the sources of neural cells for transplantation are rapidly expanding beyond the use of fetal CNS tissue and now include pluripotent ES and iPS cells as well as cells obtained by direct reprogramming. These new options can provide considerable advantages with respect to preparation of cell stocks and the use of autologous grafting, but they present challenges of complex differentiation protocols and risks of tumor formation. It is important to note that although neural stem cell transplantation into the injured spinal cord is primarily designed to provide preclinical data for the potential treatment of patients with SCI, it can also be used to develop analogous protocols for repair of neuronal circuits in other regions of the CNS damaged by injury or neurodegeneration. The advantages of the spinal cord system include well-defined structures and a large array of quantitative functional tests. Therefore, studying the formation of a functional relay will address the fundamental aspects of neuronal cell replacement without the additional complexities associated with brain circuits.
Traumatic spinal cord injury (SCI) affects approximately 17,000 people every year in the United States and is caused primarily by vehicle crashes, sport-related injuries, violence affecting mostly young men, and a growing population of the elderly suffering falls (see). There are no effective treatments for SCI, and current clinical interventions consist of operative decompression, stabilization of the lesion area, management of secondary complications, and rehabilitation (Tator, 2006). The pathophysiology of SCI is complex and begins with acute primary damage, which is followed by a secondary stage of injury characterized by a complex set of events, including cell death, ischemia, demyelination, inflammation, and scar formation (Rowland et al., 2008). Importantly, the injury disrupts the connectivity of descending and ascending tracts between the brain and the periphery, as well as spinal circuits, with little or no spontaneous regeneration, resulting in deficits in motor, sensory and autonomic function (Becker & McDonald, 2012). The poor regenerative ability of CNS axons has been studied extensively and reflects the limitations of their intrinsic growth capacity and the inhibitory environment of the injury (Cregg et al., 2014; Dell’Anno & Strittmatter, 2016). The complex and multifaceted nature of SCI, which evolves temporally and spatially, presents therapeutic challenges but also provides opportunities for innovative approaches in a system that is well defined anatomically and functionally. Indeed, over the last 15 years, investigators have developed a rich variety of injury models, functional outcome tests, and experimental strategies, including drug delivery, gene therapy, cell transplantation, electrical stimulation, rehabilitation, and robotics based on a brain–machine interface (Ramer et al., 2014).
Briefly presented here are the pathophysiology of SCI, the type of injury models used with rodent models, and the various therapeutic approaches that have been experimentally developed to promote repair and recovery. The review focuses on strategies that target regeneration and connectivity in the context of neural cell transplantation, with an emphasis on emerging developments in basic research and the clinic. In particular, the discussion takes in the challenges of neuronal cell replacement in experiments that are designed to form functional relays and restore connectivity.
The primary injury results in cell death associated with mechanical damage, excitotoxicity associated with leakage of glutamate from astrocytes, generation of free radicals from dysfunctional mitochondria, ischemia associated with the disruption of vascular supply, and invasion of inflammatory cells associated with the interruption of the blood–spinal cord barrier (Figure 1). Hemorrhage and progressive edema are the hallmarks of the postinjury environment, as well as interruption of connectivity with the brain, and demyelination of spared axons resulting in varied degrees of paralysis. In subsequent stages of the secondary injury, the inflammatory process continues with recruitment of microglia, macrophages, and leukocytes, infiltration of fibroblasts, and reactive astrocytes, resulting in a fibrotic scar, cystic cavities, and axonal dieback. This process continues for weeks and months with limited spontaneous repair, which is exacerbated by the development of pain and autonomic dysreflexia (Tator, 1995; Witiw & Fehlings, 2015). Given the complex and progressive cascade of SCI, therapeutic approaches can be divided into neuroprotective and neuroregenerative strategies, with the former addressing the early stages after injury to control the expanding damage and the latter addressing the restoration of connectivity and function, including cell therapy, which is the focus of this review.
SCI: Models of Injury
Multiple animal models of SCI have been developed, including laceration such as hemisection or transection, and compression such as contusion in closed and open injuries (Bonner & Fischer, 2013; Steward & Willenberg, 2017). These models have been used to evaluate specific experimental protocols, to test therapeutic options, and to serve as relevant clinical models. Thus, the choices of SCI models when studying regeneration and connectivity are often different from models used for studying neuroprotection and recovery of function. They are often designed to target specific sensory or motor pathways, but they require careful considerations for analysis that will confirm complete axotomy and provide conclusive evidence of regeneration without sparing. At the anatomical level, the use of tracers and viral vectors injected into the appropriate location allows the tracking of growing axons, determines the distance of growth following the injury and the interaction with the host environment at the injury and beyond, and identifies synaptic connections. At the functional level, it is critical to define the specificity and degree of impairment to allow assessment of recovery using behavioral tests.
For example, in studying regeneration of sensory axons, a dorsal hemisection model is used to axotomize ascending dorsal column axons from the dorsal root ganglion (DRG). At the same time, it is also important to consider the damage to adjacent tracts such as the corticospinal tract. The lesion can be performed with a knife cut or aspiration, which affects the size of the cavity and the extent of the glial scar. Injection of cholera toxin B unit (CTB), an anterograde tracer, into the sciatic nerve enables one to follow sensory axon regeneration, ideally all the way to the target dorsal column nuclei (Bonner et al., 2011). Functional deficits and recovery are more difficult to assess conclusively in this injury model because of the damage to other tracts. Another example related to the study of recovery of the motor system focuses on the corticospinal tract (CST) where the vast majority (>95%) of the axons are located in the dorsal column in rats. Typically, a tracer biotinylated dextran amine (BDA) is injected into the sensorimotor cortex, and the anatomical analysis is performed, with special attention to the issue of sparring and collateral sprouting (Weidner et al., 2001). When the dorsal hemisection is made at the thoracic levels, functional assessment of CST regeneration is focused on hindlimb activity using the BBB/BMS scales (Basso et al., 1995) or kinematics, but the functional relationship with the CST is in most cases indirect as other brainstem pathways (which are not injured) are also involved in locomotion. When dorsal hemisections are done at cervical levels to disrupt the CST, the recovery is focused on forelimb motor function related to hand function and gripping using a battery of tests that include pellet reaching, the staircase task, grip strength, and the cylinder task (Steward & Willenberg, 2017). Some of these tests can also be used for sensory recovery related to forelimb function, such as a detailed analysis of the pellet-reaching test with variations on the texture of the pellets. Because of the rich anatomical and functional assays available, the dorsal column injury has frequently been used in transplantation experiments with neural stem cells designed to achieve connectivity by formation of functional relays (Bonner & Steward, 2015).
Although complete injury in people is rare, one could argue for the experimental advantages of a complete transection because it guarantees that all ascending and descending pathways are axotomized without the need to worry about spared axons. The challenges here are to verify the completeness of the transection and to deal with the effects of body movements on the retraction of the rostral and caudal stumps. Anatomical and functional analysis indicates that there is no regeneration of axons across the gap without intervention(s), forming an effective system to test connectivity following cell transplantation, as demonstrated in recent studies (Lu et al., 2012; Medalha et al., 2014; Sharp et al., 2014).
SCI: Therapeutic Approaches
The clinical management of SCI centers on surgical decompression, but the time of intervention has been debated for a long time, ranging from recommendations for conservative to aggressive surgery (Ahuja et al., 2016). Recent clinical studies have favored early surgical intervention in a variety of spinal injuries and syndromes for a better improvement of neurological outcome associated with secondary injury. These studies help guide decision making for clinicians, but the limited improvement underscores the need for the development of effective treatments that address the pathophysiology of the injury with respect to neuroprotection, cell replacement, and promotion of connectivity (Wilson et al., 2012; Batchelor et al., 2013; O’Boynick et al., 2014; Anderson et al., 2015). In this context, it is noteworthy to mention the use of methylprednisolone, which was the first drug tested on a large scale but was eventually found to have possible detrimental effects, with little benefit (Lammertse, 2013). As a result, it was removed from the recommendation for standard of care in most countries. Consistently applied to patients with SCI are a variety of rehabilitation protocols, which are likely to be included in combination with any future therapies. Other promising therapeutic directions, which are moving into clinical testing but are not discussed here, include therapeutic hypothermia (Dietrich, 2012), epidural stimulation (Harkema et al., 2011; Rejc et al., 2015), and spinal cord stimulation (Mondello et al., 2014).
In contrast to the limited options for clinical interventions, there has been enormous growth in SCI research aimed at developing therapeutic strategies at the preclinical level using animal models of injury (Ramer et al., 2014; Siddiqui et al., 2015; Dell’Anno & Strittmatter, 2016). This review will briefly summarize the preclinical progress with a focus on cell transplantation. In general, such therapies are directed to address specific processes associated with the pathophysiology of SCI such as excitotoxicity, apoptosis, neuroinflammation, demyelination, and/or the lack of a regenerative response (Dent et al., 1996; Plunet et al., 2002). Some of the promising treatments that are currently being investigated include riluzole and minocycline, target neuroprotection. Others treatments use chondroitinase (to digest scar), inhibitors of Nogo (to reduce myelin inhibition), and Rho pathway to promote neuroregeneration (Siddiqui et al., 2015; Ahuja et al., 2017). Finally, there has been a growing emphasis on cell therapies, including non-neural treatments, such as mesenchymal stromal cells and macrophages, and neural cells, such as Schwann cells, olfactory ensheathing cells (OECs), embryonic stem (ES), and induced pluripotent (iPS) cells (Schwartz & Yoles, 2005; Oliveri et al., 2014; Roet & Verhaagen, 2014; Bonner & Steward, 2015; Kanno et al., 2015). It is important to note that the complexity of the multifactorial progression of SCI is likely to require a combinatorial approach that will include components of drug delivery, cell transplantation, and rehabilitation strategies.
Cell Therapy in SCI
Type of Cells for Transplantation
In general, cell therapy approaches can be divided non-neural sources and neural cells, which are the focus of this review. Additional resources describing the properties of the cells and their application in cell transplantation experiments are provided in the following references: Zhang et al. (2012); Tabakow et al. (2013); Ekberg and St John (2014); Tabakow et al. (2014); Deng et al. (2015); Gladwin and Choi (2015); Kanno et al. (2015); and Wu et al. (2015).
Non-neural cells: A variety of mesenchymal stem cells (MSCs) have been extensively used experimentally and clinically for therapy in SCI (Cao & Whittemore, 2012; Oliveri et al., 2014; Oh et al., 2016). Although their phenotypic potential is within the stromal lineage (e.g., osteoblasts, chondrocytes, adipocytes), they have a wide range of therapeutic properties and some practical advantages. MSCs have been found to secrete therapeutic factors that promote neuroprotection and axon growth, and to modulate the immune response that promotes tissue sparing. The ability to derive MSCs from a variety of sources (e.g., bone marrow, adipose tissue, placenta, amniotic fluid, and umbilical cord) allows a relatively easy preparation of a large number of autologous cells for transplantation, an advantage that is further enhanced by the ability to effectively deliver these cells via intravenous or intrathecal infusion. Indeed, there are numerous clinical trials using MSCs for SCI, some of which are reaching phase III (Oh et al., 2016).
Neural cells: Neural cells tested experimentally for SCI therapy are derived from either peripheral tissue sources (OECs and Schwann cells) or a variety of CNS neural stem cells (NSC) and progenitors with a potential to generate neurons, oligodendrocytes, and/or astrocytes. Again, both OECs (Transplantation of Autologous Olfactory Ensheathing Cells in Complete Human Spinal Cord Injury, Clinicaltrials.gov NCT01231893) and Schwann cells (Safety of Autologous Human Schwann Cells in Subjects with Subacute SCI, Clinicaltrials.gov NCT01739023) are being tested in registered clinical trials. NSCs or neural progenitor cells have been isolated from fetal tissue either as multipotent cells (neuroepithelial) or as neuronal/glial restricted progenitors (NRP/GRP). (Bonner et al., 2013). However, NSC can also be prepared from pluripotent cells such as embryonic stem (ES cells) (McDonald et al., 1999) and induced pluripotent stem (iPS) cells (Nutt et al., 2013), with the distinct advantage of an unlimited supply of cells and potential for autologous cells (for iPS cells). The complexity of deriving pluripotent cells followed by complex protocols of differentiation combined with the risk of tumor formation have recently been circumvented by the ability of direct reprogramming of adult cells (such as donor fibroblasts) into induced neurons (iN) (Thier et al., 2012).
Protocols and Analysis of Cell Transplantation in SCI
Cell delivery: To achieve effective transplantation, it is desirable to minimize the invasiveness of the procedure. Direct delivery of cells into and around the injury will cause additional injury and inflammation, which may affect the survival and phenotype of the transplant. To reduce the potential damage, transplantation is carried out using fine needles and a slow injection of the cells, but alternative methods have been developed for intrathecal and intravenous transplantation protocols, which have been implemented in clinical trials mostly with mesenchymal cells (Oh et al., 2016).
Time of transplantation: Another consideration in the design of transplantation protocols in SCI is whether to use acute/subacute or chronic injury models. The very early stages of the injury present challenges related to transplant survival because of the toxic environment of the injury, and they are also unrealistic clinically because of practical and risk issues. Thus, both preclinical experiments and clinical protocols have focused on subacute transplantation, which defines a therapeutic window of effectiveness. In contrast, transplantation in chronic SCI has not received enough attention, despite representing the vast majority of patients, because it is generally assumed that it is best to test promising therapies first in subacute injuries, where the potential for repair and recovery is higher (Tetzlaff et al., 2011).
Immune suppression: There are three types of experimental transplantation protocols with respect to the identity of the cells: isogeneic (autologous), allogeneic, and xenotransplantation. The most common protocol for either preclinical or clinical transplantation is to use allogenic cells, which requires immune suppression, but provides considerable experimental and practical advantages. For example, large stocks of cells can be characterized (and get FDA approval), aliquoted into frozen stocks and used in multiple transplantation sequences (patients). This is the model used for clinical trials involving oligodendrocyte progenitor cells (OPCs) transplantation initiated by Geron and continued by Asterias (Priest et al., 2015). Allogeneic transplantation however, requires the use of immune-suppressants such as cyclosporin or FK506 (individually or in a cocktail) to support survival of transplants. The challenge for NSC transplants used for cell replacement is that immune-suppression needs to continue indefinitely, increasing immunological vulnerability for opportunistic infections and tumor formation. Preclinical rodent models used for testing human cells are categorized as xenotransplantation and therefore need aggressive immune-suppression protocols using high doses and combinations of drugs (Yan et al., 2006; Sontag et al., 2013). Alternatively, it is possible to use immunodeficient animal models, including athymic rats and NOD-SCID mice, which minimize the risk of immune rejection but impose extra cost and special housing (Salazar et al., 2010; Jin et al., 2011). Autologous or syngeneic transplantation is another way to circumvent the immune-suppression obstacles in both experimental and clinical situations. Autologous transplantation has been used extensively with MSCs and Schwann cells, but with the development of the iPS and reprogramming technologies, they could also be applied to produce autologous NSCs to generate neurons for connectivity, oligodendrocytes for myelination, and astrocytes for neuroprotection.
Scaffolds: Biomaterials have been used extensively as a multifunctional strategy to provide a permissive matrix at the site of injury, promote survival and integration of transplants embedded into the scaffold, and deliver therapeutic factors by controlled release. Biomaterials can be polymeric, designed as nano/microspheres, or hydrogels. They can be performed or spun as a directional scaffold possibly with channels for alignment of axons or blood vessels, used as an injectable hydrogel with a phase transition at body temperature, and made to be either biodegradable or stable (Elliott Donaghue et al., 2014; Iyer et al., 2016; Raspa et al., 2016). Besides a mechanic support system, biomaterials alone can provide a local bridge to inhibit scarring and facilitate axon and blood vessel growth in an aligned manner (Tsintou et al., 2015). Hydrogels usually have three-dimensional porous structure, which permits the ingrowth of supportive tissue. When biomaterials are combined with other regenerative strategies, such as active peptides, growth factors, and stem cells, they may induce dramatic effects on neural regeneration after SCI (Krishna et al., 2013). The vast possibilities offered by material engineering create both opportunities and challenges for cell transplantations. The opportunities of collaborative work in fields such neuroscience, stem cell biology, and biomedical engineering translate into experimental design that can target multiple functional targets and precise matching of the cells and host environment. However, the complexity of the injury environment in the context of a contusion injury, the inflammatory response, and the variety of cells present at the transplantation site mean that harnessing the full beneficial potential of scaffold is difficult. For example, the use of an aligned scaffold can be nicely demonstrated in hemisection models that generate a distinct cavity where the scaffold can be properly oriented, but not in a contusion injury where the cavity may form in an unpredictable way. Interestingly, a scaffold can be used in vitro to study the intrinsic properties of neural stem cells (differentiation, migration) as well as their interactions with other cells (endothelial, immune cells) and bioactive molecules (neurotrophin gradients, inhibitory chondroitin sulfate proteoglycans) in a three-dimensional matrix.
An example is a recent study combining stem cell transplantation with biomaterial scaffolds, which provided potential advantages of enhanced cell survival, greater transplant retention, reduced scarring, and improved integration at the transplant/host interface (McCreedy et al., 2014). Using a protocol for enriching mouse ES cell-derived progenitor motor neurons (pMNs), the study demonstrated the survival and differentiation of the enriched pMNs within a 3D fibrin scaffolds in vitro, followed by transplantation into a subacute dorsal hemisection model of SCI where the cells differentiated into neurons, oligodendrocytes, and astrocytes (McCreedy et al., 2014). In another recent study, mouse embryonic fibroblasts were reprogrammed into induced neural stem cells (iNSCs), embedded into a 3D electrospun PLGA-PEG nanofiber scaffold, and transplanted into transected rat spinal cords. The analysis showed iNSC survival and differentiation into neural cells within the scaffolds, restoring continuity of the spinal cord and reducing cavity formation. This strategy also contributed to modest functional recovery of the spinal cord (Liu et al., 2015).
Analysis of transplants: The analysis of the transplant includes cell survival, phenotypes, migration, and integration, which can be facilitated by specific cell labeling (Bonner & Fischer, 2013). Effective labeling is accomplished by use of transgenic animals or cell labeling with plasmids or viral vectors. Transgenic mice and rats expressing green fluorescent protein (GFP) or human placental alkaline phosphatase (AP) have been successfully used to prepare cells for transplantation, which can be identified and followed when transplanted into the spinal cord. Similarly, mouse genetics or vectors driven by specific promoters can also be used to label specific populations of cells. Labeling cells with ferromagnetic particles (or related methods) and the subsequent tracing with magnetic resonance imaging (MRI) permits noninvasive tracking of cells after transplantation (Zhang et al., 2004; Sykova & Jendelova, 2007). Bioluminescence labeling also can be used to follow the transplant in a continuous and noninvasive protocol. When using human cells, there are markers such as human specific nuclear antigen, mitochondrial antigen, nestin, and glial fibrillary acidic protein (GFAP) that can identify the transplanted cells and their phenotype. Also, because human cells are extremely sensitive to diphtheria toxin compared to rodent cells, they can be selectively ablated when testing for their potential functional role in the transplantation (Hooshmand et al., 2009).
Neural Cell Transplantation in SCI
Advantages and Challenges
Transplantation of neural cells following SCI has the unique potential to provide the cellular components to replace neural cell lost or damage which, depending on their lineage, may include neurons, oligodendrocytes, and astrocytes (Mothe & Tator, 2013). They can also provide additional benefits targeting multiple elements of the injury by producing growth factors to support neuroprotection, by modulating neuroinflammation, by inducing remyelination, and by promoting connectivity of host regeneration as well as formation of relays. Neural cell transplantation also faces unique challenges regarding its effectiveness and safety. It requires careful selection and isolation of appropriate cells with respect to their lineage properties, followed by expansion to produce large and uniform population of cells without risking tumor formation when derived from pluripotent cells (ES and iPS cells) as shown in Figure 2. Because transplantation of neural cells is often designed for cell replacement, it is important to assure long-term integration and functional maturation (e.g., neurons for relays or oligodendrocytes for myelination) (Lane et al., 2016). In the case of a neuronal replacement strategy and formation of functional relays, it requires experimental solutions to resolve additional challenges associated with selecting appropriate regional phenotypes and functional synaptic connections, and overcoming the inhibitory injury environment to allow for connectivity. There are also experimental considerations concerning the timing and the method of cell delivery, the need for immune-suppression in allogeneic transplantation, and the development of procedures for monitoring the transplanted cells and analyzing functional outcome. Altogether, the protocols for neural cell transplantation are developed in reference to acute, subacute, and chronic injuries; to therapeutic targets such as neuroprotection, myelination, or regeneration; and as a strategy to alleviate specific functional issues such as pain, respiration, or autonomic function.
Unlike their adult counterparts, immature neurons have an intrinsic capability to extend axons and connect with host neurons when transplanted into the adult injured spinal cord. They can directly differentiate into distinct types of neurons and replace damaged or lost cells, project axons to target neurons in the cord, and support the growth and regeneration of damaged host axons into the transplant. Thus, these grafted neurons can serve as a substitute to relay supraspinal and local neuronal connections across the lesion, thereby reestablishing neuronal pathways for functional recovery (Figure 3). Early studies using embryonic spinal cord tissue transplanted into the adult rat injured spinal cord (Reier et al., 1986) demonstrated that acute transplants in either incomplete transected or contusion models achieved a high rate of survival and reliable neuronal differentiation and partial integration with the host spinal cord. Axonal projections from the transplants were visualized with anterograde tract tracing. In a later study (Jakeman & Reier, 1991), axons originating from the transplants were identified with both anterograde and retrograde tracers showing elaborate axon terminals surrounding host neurons, which extended up to 5 mm in the host cord. Although some host fibers were seen extending into the transplants, the majority of the labeled axons formed terminal-like profiles within 0.5 mm of the host-graft interface. Similarly, when cells were transplanted into the injured cord in the chronic stage, some propriospinal neurons and brainstem serotonergic neurons projected axons into the graft traversing interfaces where the dense glial scar formed along the walls of the chronic lesion site was lacking (Houle & Reier, 1988). Another early study tested the capability of axon growth from transplants in naive CNS (Li & Raisman, 1993). Mouse embryonic hippocampal neurons were transplanted into the cervical dorsal column region of adult rat spinal cord where corticospinal tracts and the ascending sensory pathways are located. Using a mouse-specific marker, the study reported that the transplanted neurons grew long-distance axons in both rostral and caudal direction within the tract. The donor axons were intermingled with the host myelinated axons. Although these pioneering studies faced technical limitations with respect to transplant labeling and axon tracing, they provided an important framework and guidance for subsequent experiments that exploited the increased understanding of the properties of neural stem cells and the availability of transgenic animals.
Defined Populations of Cells
Target: Neurons for Formation of Relays
Taking advantage of transgenic techniques, intrinsic cellular markers, and viral reporters, new strategies have been developed to overcome the difficulty of transplant visualization by using GFP and AP genes to identify transplanted neurons. Early transplantation experiments into the uninjured spinal cord using multipotential neural stem cells have shown that the grafted cells were restricted to the glial lineage because of the limitation of the non-neurogenic environment (Cao et al., 2001). In contrast, transplantation strategies using neuronal restricted precursors (NRP) derived from embryonic spinal cord of AP transgenic rats (Han et al., 2002; Lepore et al., 2004; Lepore et al., 2006) showed that the grafted NRP could survive and integrate with both gray and white matter, elaborate long processes, migrate selectively along white matter, and express markers of differentiated neurons but not of astrocytes or oligodendrocytes. These NRP also expressed specific neurotransmitter phenotypes and synaptophysin, suggesting synapse formation between transplant and host neurons. The results underscored the advantages of NRP in their commitment to a neuronal phenotype and indicated that the adult CNS allows the survival, maturation, and integration of NRP cells, circumventing the limitation of the non-neurogenic environment. However, when NRP cells were transplanted into the injured spinal cord, they survived poorly and their neuronal differentiation seemed to be inhibited, with most of the cells expressing immature neuronal markers (Cao et al., 2002). To facilitate survival and neuronal differentiation, NRP were co-transplanted with glial restricted precursors (GRP), which generate supportive astrocytes, secrete trophic factors and extracellular matrix at the lesion site, resembling the permissive environment during development (Lepore & Fischer, 2005). In the co-transplantation experiments, the NRP survived and differentiated into neurons expressing mature neuronal markers, including glutamatergic phenotypes, confirming that the GRP provide a microenvironment to protect and instruct transplanted NRP differentiation into neurons. Interestingly, the transplanted GRP could migrate out of the lesion site along white matter tract, whereas the NRP remained in the injury site. The combinatorial transplantation strategy of NRP/GRP was not sufficient for graft-derived neurons to extend axons into the host spinal cord unless a gradient of neurotrophins was produced by injection of a lentivirus expressing brain-derived neurotrophic factor (BDNF) adjacent to the transplant. Such a gradient stimulated directional outgrowth from a dorsal column lesion, establishing the necessary conditions for creation of a relay (Bonner et al., 2010).
Subsequent experiments combined transplantation of NRP/GRP into a dorsal column lesion, with injection of BDNF-expressing lentivirus into the dorsal column nuclei (DCN) to guide graft axons to the appropriate target (Bonner et al., 2011). Analysis of anterogradely traced sensory axons showed that they regenerated into the transplant, while robust graft-derived axons grew along the neurotrophin gradient into the DCN. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed functional synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN, with the predicted delay resulting from replacing a monosynaptic connection by a relay. Since BDNF also increases cell excitability and strengthens newly formed synapses, particularly in nociceptive pathways, this may play a role in the functional recovery (Pezet et al., 2002; Boyce & Mendell, 2014).
Compared to contusive or other incomplete surgical injuries, a severe SCI, such as a complete transection, presents a formidable challenge for the survival and integration of transplants with the host tissue. Under these conditions, the main experimental difficulties are the physical separation of the spinal cord and the harsh local environment at the lesion site. Specifically, the inflammatory response at the acute stage induces secondary tissue damage, which is detrimental for transplant survival. In the chronic stage of the injury, both glial and fibrotic scars form a physical and chemical barrier for axon regeneration. To address these difficulties, fetal spinal cord cells were transplanted into the subacute lesion of a completely transected rat adult spinal cord (Lu et al., 2012, 2014). E14 spinal cord cells were freshly dissected from GFP transgenic rats, dissociated into single cells, and embedded into fibrin matrices containing a cocktail of growth factors, and then grafted into the lesion site of a completely transected adult spinal cord. These cells were not cultured and therefore represented embryonic spinal cord grafts composed of a mixture of neural stem cells, mostly NRP and GRP (Kalyani & Rao, 1998), but may have included some endothelial cells and fibroblasts. These grafts differentiated into mature neurons, extended numerous axons over a remarkable distance, and formed abundant synapses with host neurons, leading to electrophysiologically active relays across the lesion. However, a replication study by the Steward group could not replicate the functional improvement results because in some of the animals the transplant/host interface was separated by fibrotic tissue or cysts preventing effective connectivity, and in some animals ectopic depositions of cells were found at long distance from the lesion (Sharp et al., 2014) with a response by original authors (Tuszynski et al., 2014a, 2014b). Taken together, these studies demonstrate the potential for the formation of functional relay across the injured spinal cord. These studies also underscore the remaining challenges, which include robust survival and integration of the transplant, control over the phenotype of the cells for excitatory glutamatergic neurons, directional guidance of graft-derived axons into putative targets without maladaptive plasticity, which may generate pain, and formation of mature and functional synapses as discussed in details in a recent review (Bonner & Steward, 2015).
Using similar strategies, embryonic brainstem-derived cells were implanted into a completely transected spinal cord to generate serotonergic and catecholaminergic neurons (Hou et al., 2013). The analysis showed numerous differentiated neurons with serotonin/catecholamine phenotypes in the graft, projecting axons across the lesion and innervating the caudal autonomic nuclei of the host spinal cord. In addition, supraspinal vasomotor axons regenerated into the graft, suggesting the potential for restoring supraspinal control. Indeed, cardiovascular function partially recovered according to measures of basal hemodynamics and autonomic dysreflexia. Another functional target assessed by transplantation of neural cells derived from fetal spinal cord tissue has been the respiratory system and the electrophysiological activity of the phrenic nerve following a high cervical spinal hemisection (White et al., 2010). The study used transneuronal tracing to demonstrate connectivity between donor neurons and the host phrenic circuitry as well as restored activity of the phrenic nerve. Interestingly, this study also revealed differences between cells derived from dorsal and ventral spinal cord, indicating that the outcome of transplantation experiments may depend on the properties of donor cells, underscoring the potential advantages of using defined populations of neurons. Using the same injury model, transplantation of embryonic midline brainstem (MB) cells, rich in serotonergic neurons, showed that although there was no impact on baseline breathing patterns during a brief respiratory challenge, rats with successful MB grafts had increased ventilation compared to rats with failed MB grafts. Recordings from the phrenic nerve also indicated increased output during respiratory challenge in rats with successful MB grafts (Dougherty et al., 2016). In another study, acute transplantation of Subventricular zone (SVZ)-derived neural progenitors demonstrated increased inspiratory tidal volume during hypoxic or hypercapnic respiratory challenge, with increased burst amplitude in the phrenic nerve across a wide range of conditions There was a positive correlation between peak inspiratory phrenic bursting with number of surviving cells in the transplant but no evidence for neuronal differentiation (Sandhu et al., 2017). These and other studies (Lee et al., 2014) have demonstrated that despite the encouraging results using neural cell transplants to restore connectivity and function of the respiratory system following high cervical injury, the logistics of transplantation and interpretation of results depend on variables such as the type of graft, the type of injury, location and timing of grafting, survival and phenotypes of the cells, inclusion of training, and the nature of the outcome measures of recovery.
Transplantation experiments have also been performed using human cells to prepare preclinical data with several cell types. The challenge in transplanting human cells in models of rodents involves the properties of xenografts, which need either very strong immunosuppression protocols or use of immunodeficient animals such as athymic nude mice or rats (with an inhibited immune system due to a greatly reduced number of T cells). to prevent rejection. In both cases, the experiments present confounding elements that complicate interpretation and translation of the results, but at the same time they also represent the reality of human transplants. An important early study reported that human NSC prepared from fetal brain as neurospheres (HuCNS-SC) and transplanted into a spinal cord lesion in immunodeficient mice could differentiate into neurons and form synaptic connectivity with host neurons but also produce myelinating oligodendrocytes (Cummings et al., 2005). The improvement in locomotor recovery was elegantly shown to be dependent on the engrafted cells using selective ablation by diphtheria toxin, which reversed the recovery. This and subsequent studies with these cells have led to the initiation of a clinical trial by Stem Cells Inc. (Study of Human CNS Stem Cell Transplantation in Cervical Spinal Cord Injury, NCT02163876), which has since been terminated. Neuralstem Inc. initiated another clinical trial based on transplantation experiments with cells derived from embryonic human spinal cord (Yan et al., 2006) in a Safety Study of Human Spinal Cord-derived Neural Stem Cell Transplantation for the Treatment of Chronic SCI (clinicaltrials.gov NCT01772810).
The modest progress and difficulties in initiating and sustaining clinical trials of cell transplantation in models of SCI underscore the large expenses associated with cell therapy, the complexity of preparing FDA-approved cell banks, the relatively small number of acute and subacute patients, and the ethical concerns associated with an invasive and risky procedure applied to a nonlife-threatening medical problem (Fawcett et al., 2007; Sorani et al., 2012; Elizei & Kwon, 2017). More recent studies have expanded the use of human NSCs derived from a variety of sources, including pluripotent embryonic stem (ES) and iPS cells. The cells were transplanted into the lesioned spinal cord of athymic immunodeficient rats, demonstrating that they can differentiate into neurons and extend numerous axons into the host tissue up to 9 cm in length (Lu et al., 2014). Immunohistochemical analyses by light and electron microscopy revealed synaptic connections between graft-derived axons and host neurons, indicating their potential integration. Although human NSC derived from ES and iPS cells remain important sources of transplant tissue, the ability for direct reprogramming of adult differentiated neural and non-neural cells into neurons by forced expression of a specific combination of transcription factors opens new possibilities for autologous grafting. Neurons have been generated in vitro from adult human fibroblasts with the introduction of three transcription factors, Ascl1, Brn2, and Myt1l, indicating the potential for reprogramming across germ layers (Vierbuchen et al., 2010) and more recently by in vivo conversion of astrocytes to neurons in the injured adult spinal cord using a single transcription factor SOX2 (Su et al., 2014). Future sources of human cells to be used in cellular therapy for SCI will become available with improved technology and optimized protocols generating a promising pipeline from preclinical experiments to clinical trials.
In addition to extending axons, transplants of early-stage neurons facilitate host axon regeneration into the graft/injury site, which are essential for the formation of a relay (Figure 3). For example, analysis of anterogradely traced sensory axons following transplantation of NRP/GRP showed that they regenerate into the transplant and make synaptic connection with graft-derived neurons (Bonner et al., 2011). When embryonic rat spinal cord cells or human iPSC-derived cells were transplanted into a lesion of SCI, host raphespinal axons penetrated the transplant (Lu et al., 2012, 2014). Furthermore, a recent study has shown that even corticospinal axons could regenerate into these subacute transplants and form functional synapses (Kadoya et al., 2016). Interestingly, cells differentiated into a caudalized fate (spinal cord) were more conducive for CST regeneration than the rostralized cells (brain). Surprisingly, some CST axons even regenerated across the graft and extended into the caudal host tissue. Behavioral tests showed that skilled forelimb function was improved. In contrast, a previous study reported that caudalized human iPSC-derived neural progenitor cells, which produce neurons and glia, fail to restore function in an early chronic spinal cord injury model (Nutt et al., 2013). Though multiple differences were involved between these two studies, such as cell sources, injury models, and forelimb/hindlimb behavioral tests, the main reason for the discrepancy might be transplantation timing after SCI regarding severe scarring in the chronic phase, a physical and chemical barrier to inhibit host/grafted axon growth. Overall, transplanted early-stage neurons not only project axonal extensions to host spinal cord neurons, but also attract host descending neuronal pathways to grow in, indicating the potential for a relay mechanism to restore neuronal signal transmission in the severely injured spinal cord.
Despite ongoing efforts and the need for further improvements, it is a fair assessment that the development of NSC transplantation strategies in SCI has reached the point that it is possible to achieve neuronal survival and differentiation in both sensory and motor systems with a variety of cells. The focus is therefore shifting to the task of designing a functional relay formed by active synaptic connections with appropriate targets, and the transformation of the restored communication by the relay into meaningful behavioral improvement. Specifically, some of the significant challenges include (1) how to select for the most effective neuronal phenotypes in building a relay, (2) how to guide graft-derived axons toward the correct target neurons while minimizing the effects of maladaptive connections, (3) how to form mature and stable synapses, and (4) how to transform the new connectivity, which is quantitatively and qualitatively different from the original innervation (e.g., fewer axons, imprecise mapping), into useful function in a system that has already been reorganized in response to lack of connectivity (Bonner & Steward, 2015). These are daunting challenges that we may be addressed by considering the process of neural development and the remarkable plasticity associated with activity and exercise. For example, the properties of the transplanted neurons during their differentiation and integration may mimic the process of neural development in which excessive synaptic connections are initially formed and then pruned by specific activity-related cues allowing the selective formation of functional synapses (Nikolaev et al., 2009). Similarly, to strengthen functional connectivity, it may be possible to apply specific training protocols to select and strengthen synapses associated with appropriate targets and desired activity. For example, specific forelimb activity protocols may reinforce grasping behavior associated with the cortical spinal tract, and intermittent hypoxia may reinforce respiratory function by strengthening synapses associated with the specific activity and eliminating synapses associated with misconnections. This process initially occurs by restoring electrophysiological activity, but it can then propagate and translate into the behavioral levels by adaptation where the activity of newly established neuronal circuits triggers reorganization of the motor and sensory cortex. The mapping generated by the restored connectivity will integrate the afferent signal and the efferent command, thereby rebuilding a meaningful neuronal circuitry. Whether such a strategy, initiated by one type of behavior training, can generalize to other activities is still not clear.
Target: Oligodendrocytes to Promote Myelination
Widespread axon demyelination occurs as a result of prolonged and dispersed oligodendrocyte cell death following SCI. Trauma-initiated degenerative cascades of inflammation and immune response often render the loss of oligodendrocyte and myelin in the vicinity of lesion as well as spared tissues. Consequently, action potential propagation is interrupted due to the exposure of voltage-gated potassium channels at the internodes. In parallel to the loss of oligodendrocytes, there is a rapid and significant proliferation and migration of progenitor cells, which differentiate into myelinating oligodendrocytes (Almad et al., 2011). Although the resulting myelination process is robust, the endogenous response is insufficient relative to the loss of oligodendrocytes and demyelination. Thus, replacing myelin-forming cells in the injured spinal cord is necessary to remyelinate injured axons, enhance action potential conduction, and improve functional recovery. This strategy, with either transplanting OPCs or mobilizing endogenous progenitors, holds great promise as a therapeutic approach for SCI (Goldman & Kuypers, 2015). As cell transplantation is now entering clinical trials for treatment of SCI first by Geron and now by Asterias (Dose Escalation Study of AST-OPC1 in Spinal Cord Injury, NCT02302157), it is necessary to ascertain whether accelerating remyelination is a valid target to restore impaired function (Plemel et al., 2014).
For transplantation experiments, a homogeneous culture of OPCs was generated from NPCs isolated from adult rat subependymal tissues. These OPs produced robust myelin after being transplanted into the spinal cord of postnatal day 6–8 myelin-deficient rats, suggesting the potential for repairing myelin disorders in adulthood (Zhang et al., 1999). Several classes of glial precursors have been used for cell transplantation into the spinal cord. NPCs derived from adult mouse brain were transplanted into the lesion site of clip compression SCI (Karimi-Abdolrezaee et al., 2006, 2010). With further support from growth factors, chondroitinase, and inhibition of microgliosis, these cells survived and differentiated into oligodendrocytes to myelinate axons, in which nodes of Ranvier formed and axonal conduction velocity was improved, leading to functional repair. This improvement is likely attributed to multiple mechanisms, including oligodendrocyte remyelination, reduced axonal dieback, and intraspinal plasticity. OPCs have been demonstrated to remyelinate denuded host axons after differentiating into myelin-producing oligodendrocytes. Adult rat OPCs were engineered to express ciliary neurotrophic factor (CNTF), the oligodendrocyte differentiation and survival factor (Cao et al., 2010). When these cells were transplanted into the spinal cord lesion nine days postthoracic contusion, they displayed better survival, enhanced OPC-derived oligodendrocyte differentiation and myelination in comparison to unmodified OPC transplants. Moreover, this transplantation partially restored electrophysiological conduction through denuded axons to promote functional improvement.
To be clinically relevant, human ESCs may be a suitable candidate to target oligodendrocyte replacement in SCI models. Previous studies have showed that human ESCs can be used to generate OPCs capable of myelination (Faulkner & Keirstead, 2005). These cells induced extensive remyelination and promoted functional recovery when transplanted into an injured rat spinal cord at the subacute stage, whereas no therapeutic efficacy was observed in a chronic lesion setting (Keirstead et al., 2005). A follow-up study examined these cells after transplantation into a subacute cervically injured spinal cord (Sharp et al., 2010). The human ESC-derived OPC transplants attenuated lesion pathogenesis and improved recovery of forelimb function. Histologically, dramatic white and gray matter sparing was observed at the injury epicenter. In addition, the preservation of motor neurons was noticed, correlated with movement recovery. Despite the functional improvement seemingly mediated via a nonremyelination mechanism, this cell transplantation strategy is a potential therapeutic treatment for SCI. This study and others demonstrate that the critical therapeutic targets of the various NPCs are difficult to interpret because the injured environment niche affects the properties of the grafted cells (Sontag et al., 2014) and because the cells can interact with the injured host in a complex cascade of events, where a direct relationship between the observed anatomical and molecular changes and functional outcome is not obvious. Nevertheless, the required preclinical trial is focused on safety and efficacy even when the mechanisms are not entirely clear. For example, when human ESC-derived OPCs were transplanted in rodent models of thoracic SCI, they promoted neurite outgrowth in vitro and myelination in vivo without any adverse clinical observations, that is, toxicities, allodynia, or tumors (Priest et al., 2015). Thus, a Phase I clinical trial in patients with sensorimotor complete thoracic SCI has been initiated, even though similar studies lack consistency and replication with respect to the progression of myelinating cell transplantation therapies into the clinic (Myers et al., 2016).
Target: Astrocytes to Promote Neuroprotection and Neuroregeneration
During CNS development, astrocytes generate a supportive environment for neurogenesis and synaptogenesis, and later they play a role in maintaining homeostasis, building the blood-brain barrier, and regulating synaptic transmission (Sloan & Barres, 2014). These permissive properties can be exploited by generating immature astrocytes from GRP intended to improve the injury environment as well as stimulate axon growth and synapse formation (Falnikar et al., 2015). Indeed, transplantation of GRP has been used in experimental SCI as a therapeutic platform to improve neuronal survival (Lepore & Fischer, 2005), stimulate axon growth and regeneration (Hill et al., 2004; Haas et al., 2012), and provide neuroprotection (Li et al., 2015). Other studies have been focused on enhancing the migration of GRP out of the injury to generate a trail for axonal guidance (Yuan et al., 2016) and on restoring the expression of glutamate transporter in order to reduce neuropathic pain (Falnikar et al., 2016). Interestingly, early studies using transplantation of multipotent NSC into models of SCI reported that they differentiated predominantly into astrocytes (Cao et al., 2001), indicating that the non-neurogenic adult CNS, in particular, the site of injury, lacks the developmental cues necessary for the formation of neurons. As GRP can produce both astrocytes and oligodendrocyte, a pre-differentiation step to produce astrocytes (termed GRP-derived astrocytes, or GDAs) was added using BMP-4 or CNTF. Indeed, the transplantation of these GDAs was shown to promote axon growth with both rodent and human cells (Haas et al., 2012; Haas & Fischer, 2013). However, there were phenotypic differences between the different pre-differentiation protocols. For example, GDAs prepared by BMP-4 treatment produced heterogeneous populations of astrocytes, whereas CNTF produced less mature astrocytes. The dynamic nature of the astrocytes and their phenotypic heterogeneity may also account for the discrepancies observed in transplantation experiments by different groups (Noble et al., 2011; Hayakawa et al., 2016).
The sources of GRP have evolved with the growing knowledge of neural stem cell biology from the early transplantation studies, which used mostly fetal tissue, to the present time where GRP can be produced from a variety of neural stem cells and progenitors, including pluripotent ES and iPS cells. Similar advances have occurred as the transplantation strategies, which were limited to rodent cells, began using clinically relevant human cell lines. The use of human cells has been facilitated by the development of efficient differentiation protocols toward the astrocyte lineage, which for human cells can be a long and expensive process (Chandrasekaran et al., 2016). The ability to harvest and maintain human cells has been underscored by the manufacturing advances made by Q-therapeutics (Haas & Fischer, 2013) and the availability of human astrocytes derived from different regions of the CNS or from different stem cell sources. Advances in the understanding of astrocyte heterogeneity and their complex interaction with other neural cells (Khakh & Sofroniew, 2015) have also refined our understanding of the process of astrogliosis as a strictly detrimental process, which inhibits regeneration, to a protective process that provides a barrier and prevents the expansion of the lesion (Sofroniew, 2015).
Conclusions and Future Directions
• SCI is a complex and prolonged process beginning with an acute primary damage that progresses with a cascade of secondary injury into chronic stages. It is characterized by an inflammatory response, cell death, demyelination, and limited axonal growth because of the intrinsic limitations of adult CNS neurons and the inhibitory injured environment.
• Advances in isolation and characterization of neural stem cells provide the potential for cell replacement and restoration of connectivity by transplants that can generate neurons, oligodendrocytes, and astrocytes.
• The premise of neural stem cells transplantation has been extensively tested using a variety of SCI models and experimental methods that examined various parameters such as local and systemic cell delivery, acute and delayed grafting, immune-suppression protocols, and the inclusion of multifunctional scaffolds.
• The ability to generate graft-derived neurons has been used to restore connectivity by formation of relays in sensory, motor, and autonomic systems disrupted by SCI. The challenges for the relay strategy include the selection of a population of neurons that maximizes the effective transmission of signals, long-term survival of graft-derived neurons, and promotion of axon growth into and out of the transplant to form functional synaptic connections.
• It is important to note that a successful restoration of connectivity will not have the same properties as the original innervation with respect to the number of axons or their somatotopic mapping, and that the denervated areas in the brain have reorganized to compensate for the injury-induced deficits. The restoration of connectivity will therefore have to be combined with activity-dependent plasticity mechanisms to achieve a useful functional recovery.
• Replacement of oligodendrocytes targets the remyelination process, which can be applied not only for SCI but also to a variety of demyelinating disorders including multiple sclerosis (MS). The generation of effective protocols for the isolation and purification of OPC, which are able to restore function, has advanced to ongoing clinical trials started by Geron and continuing with Asteria.
• The function of astrocytes following SCI has been a subject of some debate, as they appear both helpful and harmful depending on their specific phenotype, the environment and the injury phase. As a result, the premise of astrocyte replacement has been slow to develop and only recently gained interest as a strategy for improving the injury environment and supporting survival and neuronal differentiation of transplants. One promising strategy for generating permissive astrocytes is the use of GRP derived from fetal tissue or ES/iPS cells, which has been approved for a clinical trial in amyotrophic lateral sclerosis (ALS) patients by Q-Therapeutics.
• The use of human cells for replacement therapy presents the risk of tumorigenicity when using pluripotent cells (i.e., ES cells) and complication of xenografts, which need special considerations to minimize rejection, but they also present the promise of generating preclinical data with target cells and the potential for autologous grafting with iPS cells.
• The various cell replacement strategies have learned lessons from the field of developmental neuroscience in the use of combination neuronal and glial progenitors to produce a microenvironment conducive to cell survival and neuronal differentiation and in the exploitation of the remarkable plasticity associated with activity and exercise to prune and strengthen synaptic connections in promoting functional recovery.
• Another lesson from the wealth of neural stem cell transplantation experiments is that the therapeutic target associated with a specific phenotype (e.g., the use of OPC for remyelination) may have unexpected positive (e.g., neuroprotection) or negative (pain) effects.
• The sources of neural cells for transplantation are rapidly expanding and now include pluripotent ES cells, autologous iPS cells, and cells obtained by direct reprogramming, such as transforming reactive astrocytes into neurons.
• The injured spinal cord has proven to be a useful system in testing novel cell replacement strategies because of the well-defined anatomy of distinct ascending and descending tracts and a relatively simple circuitry and because of the richness of quantitative outcome measure associated with motor, sensory, and autonomic functions. Thus, data from SCI can be applied to other regions of the CNS damaged by injury or neurodegeneration.The measures of both the challenges and success of cell replacement in SCI is underscored by the initiation of several clinical trials, including the approval of one of the first human ES-derived cell transplantation, and at the same time the difficulties in sustaining such trials because of the complexities of cell therapies, the very high expenses, and the relatively small population of acute/subacute patients.
• Future directions may include a more effective use of large animal models for verification of data obtained with rodents, development of less invasive cell delivery and enhanced imaging techniques, more emphasis on autologous transplantation using new technologies of generating appropriate neural cells, inclusion of chronic SCI in clinical trials, and a better alignment between preclinical and clinical outcome measures.
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