Schwann cell transplantation for spinal cord injury (SCI) is at an early stage of clinical testing following preclinical development. Food and Drug Administration (FDA) approval of an investigational new drug application (IND) to undertake a Phase 1 safety and feasibility study in patients after subacute SCI was based on the following milestones: detailed characterization of manufactured human Schwann cell batches, pivotal preclinical safety studies, development of clinical cell injection methodology, and adequate outcome assessment methods to make a valid appraisal of feasibility and safety. Schwann cells are being tested for repair in the central nervous system (CNS) because they support axonal regeneration in the peripheral nervous system (PNS). Following nerve injury, Schwann cells dedifferentiate [1▪,2], secrete growth-promoting trophic molecules and axon growth-promoting extracellular matrix such as laminin, which support axonal growth cone elongation. Regenerated axons are then myelinated, restoring rapid action potential conduction and function. The biology of peripheral nerve regeneration across many species, including man, is similar and has been studied for more than a century . Experimental studies have tested whether Schwann cells would allow normally nonregenerative damaged CNS axons to regenerate within the traumatically injured spinal cord and myelinate damaged axons . Autologous transplantation of Schwann cells should not require immune suppression and eliminates the risk of transmission of undetected allograft donor abnormalities.
Few clinical trials have yet reported on the effects of transplanted Schwann cells in SCI or other potential applications such as nerve repair (Table 1[5–7]). Investigators in Iran reported two studies: the first with four patients with chronic thoracic SCI transplanted with autologous Schwann cells  followed for 1 year and a second report of 33 patients with 2-year follow-up . They described a method to cultivate human Schwann cells using initial serum starvation followed by exposure to autologous serum without growth factors , arguing that this technique may be safer than the use of artificial mitogens. A Chinese study enrolled six patients with chronic SCI and reported their results after following the patients for 5 years. In that study, the sural nerve was predegenerated by cutting it within the body a week before removing it for cell culture . This step may accelerate the rate of cell division in culture. These clinical studies found a low incidence of adverse events that could be linked to the transplants. Although the studies were not conducted under rigorous FDA oversight, the lack of reported serious adverse events in the patients is reassuring. Clinicaltrials.gov lists only one current study testing Schwann cell transplantation, NCT01739023, a Phase 1 clinical study of the ‘Safety and Tolerability of Autologous Human Schwann Cells (ahSCs) in Subjects With Subacute SCI’ with which the authors are associated.
SCHWANN CELLS AND PERIPHERAL NERVE REPAIR
Spontaneous recovery after nerve injuries, although imperfect, provides the core rationale to test Schwann cells in the CNS. Even when nerves are completely transected, the careful interposition of nerve graft segments derived from noncritical nerves can support axonal regeneration, leading to the recovery of muscle function and sensation[9–12]. In these clinical grafting procedures, the donor nerve grafts often do not match the size of the injured nerve stumps and there has been interest in using fabricated tubes with diameters similar to the injured nerve stumps, filled with cultured Schwann cells, to span the injured nerve gap. Animal experiments have demonstrated the feasibility of this approach [13,14] using human Schwann cells. The success and reproducibility of these experiments in nerves led to successful testing of tubing biomaterials and Schwann cells within the spinal cord [15,16] in complete transection models.
PERIPHERAL NERVE GRAFTS VERSUS SCHWANN CELL TRANSPLANTATION FOR CENTRAL NERVOUS SYSTEM REPAIR
Several investigators studied whether peripheral nerve grafts (PNGs) transplanted into continuity with spinal cord or brain tissues  could support CNS axonal growth. Regeneration into nerve grafts  confirmed the ability of some classes of damaged CNS axons to regenerate if the tissue environment is permissive. Grafts depleted of Schwann cells by freezing did not support CNS axonal growth, establishing that viable Schwann cells were essential . The suitability of PNGs for transplantation into damaged regions of nontransected spinal cord is limited by their structure and the need to manipulate injured spinal cord tissue to create a suitable interface with the nerve grafts. Suspensions of Schwann cells cultured from peripheral nerve biopsies can be delivered with less surgical spinal tissue manipulation and have several other advantages over the use of PNGs: Schwann cells can be highly characterized for phenotypic markers, purified to remove fibroblasts, and expanded exponentially to provide the large cell numbers that are necessary for adequate engraftment in spinal cord injuries. Transplanted Schwann cells can fill the injury region, migrate and insinuate to the unique dimensions of each injury, and form bridging tissue. Advances in tissue culture were necessary to permit the reliable derivation of human Schwann cell cultures from donor nerves [20–22]. The sural nerve is the most commonly harvested nerve from which human Schwann cells are derived because of its superficial location, adequate length, and the modest consequences of removing it .
HOW SCHWANN CELL TRANSPLANTATION DIFFERS FROM THE ENDOGENOUS SCHWANN CELL RESPONSE AFTER INJURY?
Normally, the spinal cord is segregated from the associated peripheral tissues such as the nerve roots and pia mater by the glial limiting membrane (GLM), formed by astrocyte foot-processes and extracellular matrix at the brain and spinal cord surface. Schwann cells are present at the dorsal root entry and ventral root exit spinal cord interfaces where nerve roots join the spinal cord, but they are not found within the parenchyma because of the specialized GLM in these areas [24,25]. Following various injuries including SCI, the GLM is transiently disrupted and Schwann cells spontaneously enter the spinal cord . In people, this leads to the formation of neuromatous structures within the injury site called ‘Schwannosis’ [27,28]. On the basis of the current information, it does not appear that Schwannosis has a significant role in the repair of damaged central axons. Schwannosis differs from Schwann cell neoplasms such as Schwannoma because of the presence of normally formed myelin and axons  in the former and their absence in the latter. Transplanted Schwann cells may be placed into the spinal cord at a specific location and time point after injury distinct from the endogenous Schwann cell response. The fact that no adverse effect has been attributed to naturally occurring Schwannosis is an important argument for the inherent safety of Schwann cell transplantation.
PRECLINICAL DATA SUPPORTING SCHWANN CELL TRANSPLANTATION IN HUMANS WITH SPINAL CORD INJURY
A PubMed search of animal experimental studies was performed using Endnote X5 with the search terms, ‘Schwann’, and ‘spinal cord injury’ in the abstract fields. The references positive for these two search terms (413) were screened for those in which Schwann cells were directly implanted into the spinal cord regardless of the injury model. Review articles were excluded. This reduced the total number of citations to 72. Of these studies, 16 tested transplantation of genetically unmodified Schwann cells in the most relevant injury model (contusion) as one arm in the study. To summarize the observations from these 16 studies, it was consistently found that some proportion of Schwann cells engrafted and supported axonal sprouting and myelination. None of the prior studies was performed in compliance with Good Laboratory Practice (GLP), the standard expected by the United States. FDA [30▪▪,31] and none used Schwann cells that were prepared to current Good Manufacturing Process (cGMP) standards. Therefore, the prior published studies were not suitable as pivotal studies to support an IND application.
The authors and their colleagues at the Miami Project to Cure Paralysis conducted detailed toxicity studies that were designed to support an IND application to conduct a safety study of autologous Schwann cell transplantation in subacute SCI. The IND was submitted to the FDA in September 2011 and approved in July 2012. Three animal models were used: rodents, minipigs, and primates. The rodent studies were designed for robust statistical analysis of cell survival and engraftment, whereas those in the larger animals addressed the issues related to the transplant methodology and used autologous cell preparations. Together, these studies demonstrated long-term cell survival and the absence of abnormal cellular formations throughout the brain and spinal cord.
RESEARCH DESIGNS IN CELL THERAPY CLINICAL TRIALS
Despite extensive preclinical research, the effects of a therapeutic in humans with the target disease cannot be fully predicted until rigorous clinical testing occurs with adequate long-term follow-up. Control groups are problematic in cell therapy trials because it is unrealistic to place research individuals at the risk of surgical exposure, anesthesia, and postsurgical recovery in order to perform a noncellular control injection. Another approach could be to randomize individuals to two or more treatment groups, such as two different cell types, or the combination of cell therapy plus another biological therapy versus cell therapy alone. In these situations, similar risks and the existence of equipoise could justify such research designs. However, most cell therapies for SCI are at the Phase 1 safety study stage of development, and it would be complex from a regulatory and informed consent point-of-view to perform these comparisons until clinical data regarding the individual therapies are available. Thus, if control groups in early studies are used, they are generally prospectively matched individuals assigned to the best standard care [32▪▪]. Relevant data registries can provide important comparator information for the incidence of adverse events [33▪▪] and anticipated neurological outcomes .
The first cell therapy for SCI conducted under an FDA approved IND that has been reported is the Proneuron study [32▪▪]. This Phase 2 study of autologous activated macrophage transplantation was terminated prior to full enrollment for financial reasons, as was the subsequent FDA approved Phase 1 Geron study of embryonic stem cell transplantation [35–38], indicating the difficulty of maintaining financial support of cell therapy trials in neurological diseases. Another FDA approved cell therapy study that has published safety data is the Neuralstem amyotrophic lateral sclerosis (ALS) study, in which neural stem cells are implanted [39▪▪,40] into the spinal cord of patients with advanced ALS to slow the disease progression. Cell therapies for SCI are particularly expensive because of the use of surgery and anesthesia, the need for in-hospital acute care and rehabilitation, advanced imaging, cell manufacturing costs, and extensive follow-up.
In the next section, we address the issues that have been most important to the initiation of our clinical study.
Summary of enrollment criteria for our Phase 1 safety study
The Miami Project study selects for those patients with the least risk to be harmed neurologically by the cell transplantation. Thus, patients with thoracic SCI with neurologically complete injuries are enrolled because their prospect for natural recovery is minimal . Injuries at the thoracic spinal cord level were selected because loss of function in nearby spinal segments as a complication would be less harmful than in the cervical spinal cord. Important exclusion criteria include the inability to adequately image the SCI and implantation site using MRI. The configuration of spinal fixation instrumentation  may generate MRI artifacts obscuring the injury and transplant site precluding critical safety evaluations to assess for the formation of an intraspinal mass. Another important consideration in this study is to determine that transplanted Schwann cells do not exacerbate harmful neuroplasticity. After SCI, neuropathic pain is common [42,43], and cell transplantation could theoretically exacerbate this problem . Thus, patients who develop severe neuropathic pain are excluded from transplantation.
Each autologous cell culture is unique. This poses a challenge to generate cell products that are sufficiently similar, so that their effects after transplantation may be compared. It is expected that individual variations in genotype, anatomy, and life history mean that each donor nerve is different (Table 2[5–7]). Even when carefully replicated procedures such as nerve dissection, seeding onto laminin-coated surfaces, and exposure to media components and growth factors are identical, the growth kinetics of the culture and its cellular composition may vary to some extent. In some patients, it may not be possible to obtain adequate cultures for transplantation and this must be explained during informed consent. Furthermore, assessment of the number of failed cultures compared with successful cultures is an important aspect of the determination of the feasibility of the autologous transplant program. The Schwann cell manufacturing capacity is exponential, such that millions of cells can be generated from a modest segment of donor nerve. Although Schwann cells undergo dedifferentiation in cell culture and may exhibit considerable plasticity , there is no current evidence that cell culture leads to cellular changes that impair their ability to function as myelinating and regeneration-promoting cells after implantation into the injured spinal cord (unpublished data, IND 14856; Fig. 1).
Cells are implanted into the damaged spinal cord by direct injection. There are several variables to consider in the development of a safe injection method. Transplant injections have the potential to create damage to preserved spinal tissue in several ways and associated injury must be minimized. The most damaging injections are those of large volumes, delivered rapidly, with poor control over motion of the needle and tissue interface . In the clinical environment, it is important to consider all contingencies that might add risk during the cellular transplantation because unpredictable events such as, for example, electrical power failure or anesthetic emergencies, including cardiopulmonary instability, although uncommon, do occur. Therefore, the ability to terminate the injection and exit the spinal cord rapidly is necessary. A rigid needle within spinal cord tissue can cause serious injury if there is loss of control of the position of the needle because of operator error, injection device dysfunction, or inadvertent patient motion. Currently, there are three main approaches to make spinal cord injections: free hand needle injections, fixed platform injections, and floating cannula injections [47,48]. Each method has specific merits and limitations. We currently use a fixed platform injection apparatus.
Dose of cells
Selecting the optimal clinical cell dose is a challenging task because of the complex effects of cells compared to more conventional drugs. For example, after most cell injections, some cells will die and engender some degree of inflammation. Thus, both beneficial and harmful events may occur simultaneously after transplantation. The best cell dose is a function of the final result in the tissue and may not be based solely on a single tissue effect. There is a limited ability to monitor toxicological endpoints in SCI patients receiving cell transplants other than worsening of the neurological injury density or level. Complications of SCI such as neuropathic pain and spasticity occur to some extent in most patients and linking these endpoints to the cell dose may be difficult. The formation of abnormal tissue or tumors may occur independent of the cell dose. In our IND development, we have focused on learning the maximum tolerated dose that can be delivered to the spinal cord in animals and not cause additional injury that is evident by clinical examination, neurophysiology, postinjection MRI, or histology. We found that the minipig SCI model was very useful for dose tolerance studies because of its human-like neural axis dimensions. On the basis of large animal testing, we determined a well tolerated dose at which to initiate the study and successive larger doses that may exert a superior therapeutic effect.
In our current study, the most important outcomes are the feasibility of the autologous transplant strategy and the safety of the procedures and cellular implant. Impairment of residual neurological function could occur as a result of the surgical implantation procedure or because of the biological effect of the cell transplant. In patients with complete thoracic SCI, such changes are measured using sensory testing, with the neurological level as the endpoint. This level is defined as the last at which sensory perception is normal on both sides. The outcome measures we are utilizing are listed in Table 3[5–7].
The need for surrogate markers of cell survival, engraftment, and effect
It is desirable to have clinical tests that allow the effect of cell implantation to be followed longitudinally, especially to determine cell survival and biological activity. This is important because a clear impact on neurological recovery may not occur with cell grafts alone and will likely require future combination therapies. The paucity of surrogate markers is not unique to Schwann cell transplantation, but is a general problem facing the CNS cell therapy field. The doses of transplanted cells are relatively small compared with the overall cell death that occurs after SCI, potentially masking the ability to detect Schwann-cell-specific markers of cell death and survival. For Schwann cells, the issue is further complicated because of the fact that endogenous Schwann cells enter the regions of SCI and may have similar biological activity. Because allografts require immune suppressive drugs to avoid cellular rejection, formation of antiallograft antibodies is a useful biomarker that is not available for autografts to determine a definite host response. It is likely that progress in this area will require the development of well tolerated molecular markers that the transplanted cells can uniquely express and which do not impair their biological activity in the long term.
Autologous Schwann cell transplantation is a reasonable treatment approach to the repair of spinal cord injuries based on the role of Schwann cells in peripheral nerve repair, the endogenous Schwann cell's response to spinal cord injury, and the feasibility of preparing and delivering the cells. More clinical experience is required to determine the safety and efficacy. It is probable that future studies will combine Schwann cell transplantation with additional therapies to amplify the reparative effects.
The Phase 1 Safety trial of Schwann cell transplantation for subacute spinal cord injury is supported by the Miami Project to Cure Paralysis and the Bounniconti Fund. The stereotaxic injector is supplied by Geron corporation. Dr. Ed Wirth provided valuable input regarding the use of the stereotaxic injector. Key study personnel include: Dr Allan Levi MD, PhD, Co-PI; Dr Dalton Dietrich PhD, Sponsor; Dr Kim Anderson PhD; Dr Diana Cardenas MD; Dr Mary Bunge PhD; Dr Damien Pearse PhD; Dr Pat Wood PhD; Ms Aisha Khan MBA PhD; Dr Marina Dididze MD PhD; Dr Paula Monje, PhD; and Mr Anil Lalwani, Project Manager.
Conflicts of interest
J.G. is co-PI of the study SC NCT01739023, a Phase 1 clinical study of the ‘Safety and Tolerability of Autologous Human Schwann Cells (ahSCs) in Subjects With Subacute SCI’.
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