Spinal cord injury (SCI) throughout history

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1 Overview of Cellular Therapies in Spinal Cord Injuries Jesse OWENS Biomedical Program, University of Alaska, Anchorage, Alaska Spinal cord injury (SCI) throughout history has been considered untreatable. However, in recent years a great deal of knowledge has emerged regarding the major obstacles to healing and recovery of function in chronic spinal cord injury (csci). There are both cellular and molecular barriers that impede recovery of normal function in SCI. This manuscript describes the major obstacles in SCI and suggests what role a variety of cell types may play, alone or with other therapeutic agents in treating SCI. Introduction Spinal cord injury (SCI) is a catastrophic event that leads to permanent functional deficits. The financial cost to society and the decline in the quality of life for people affected by SCI and their families are enormous. Spinal cord injury results in severe medical consequences including paralysis and loss of sensation below the level of the injury. There are serious secondary problems including bone demineralization, frequent urinary tract infections, bladder dysfunction, kidney stones, incontinence, development of contractures, spasticity, pressure sores from continuous sitting, hypotension, inappropriate bowel motility and loss of sexual function (Johnson et al, 1998, Steinberg 1980)). To compound the difficulty social isolation, depression, drug addiction, and failed interpersonal relationships often accompany SCI (Craig et al, 1994, Westgren and Levi, 1998)). As medical procedures have improved over the last few decades the number of people surviving SCI has increased dramatically, leaving an ever increasing population of people with life-long paralysis. Loss of function due to SCI has been considered to be irrevocable until recently. However, now several treatment approaches to SCI in both human and animal models have produced limited recovery of function. Much of the optimism for treating spinal cord injury derives from the discovery that various cell types can assist in healing the damaged cord, even in the chronically injured cord (Kim and Vellis 2009, Coutts and Keirstead 2008). In humans recovery of function from transplantation of cells may be limited but even partial restoration of function could yield major improvements in quality of life including improved breathing, grasp, movement in legs and arms, and perhaps control of bowel, bladder, and sexual function. While the basic science of SCI continues to unravel the recalcitrance of the damaged cord to heal many human studies are already in the works. At present many hundreds of people with SCI have been treated with various cell types at many diverse locations on earth. Unfortunately, due to the lack of scientific rigor in the majority of these studies we still can not draw confident conclusions about the relative effectiveness of specific treatments. Pathology of chronically injured spinal cord Within few weeks after the initial insult to the spinal cord the injury site is completely remodeled. The distal ends of severed axons breakdown and die (Fig. 1B). However the neuronal cell bodies and proximal axon segments may remain alive and potentially functional if it were only possible for them to grow past the injury site to their original synaptic target (Silver and Miller 2004). Astrocytes and fibroblasts produce a wall around the dam Ulusal Hematoloji Kongresi

2 Overview of Cellular Therapies in Spinal Cord Injuries OWENS J. aged region of the cord resulting in a fluid-filled cavity. The astrocytic scar produces a physical barrier to growing axons, but perhaps more importantly the scar is replete with axon-growth inhibitory molecules, primarily Chondroitin Sulfate Proteoglycans (CSPGs). Even more inhibition to axon growth is found on the surface of myelin. The major inhibitors on myelin are NogoA, Myelin- Associated Glycoprotein (MAG), and Oligodendrocyte Myelin Glycoprotein (OMG). In regions adjacent to the injury site many long-tract axons may survive intact, however oligodendrocytes providing the myelin for these axons die as a result of the trauma and ensuing inflammation, leaving the axons intact but incapable if conduction (Miller and Silver 2004)(Fig.1A). There are many different cell types that have been transplanted into humans and even more into animals. In humans a partial list includes: Bone marrow cells (BMSCs)(mesenchymal, and crude stromal cell extract), olfactory ensheathing cells (OECs), olfactory mucosa, umbilical cord cells, fetal human and pig neural stem cells (NSCs), activated macrophage and embryonic stem cells (ESCs) (Kim and Vellis 2009). A recent search of the literature by the author revealed at least 39 human trials are underway or have been recently completed. These trials involved transplantation of both allogenic and autologous cells. The variability within these human trials is so great it is difficult to make a comparison of the results. However, to my knowledge (with the exception of one Russian transplant recipient) there has been no obvious harm to patients. There appears to be some recovery of function in some of the treatments, even in chronic SCI. Major components of csci that must be addressed in an effective treatment Inhibition of axon growth from scar and myelin inhibitors Enhancement or restoration of dormant pathways by re-myelination Render the lesion Scar permissive to axon growth Replacement of lost cell types Long term drug delivery to spinal cord How do transplanted cells help resolve these impediments? Bystander effect In the spinal cord stem cells appear to exert a bystander effect when faced with injured tissue. The bystander effect refers to the transplanted stem cell producing a favorable environment for tissue repair without becoming integrated into the local population of differentiated cells (Ankey et al 2004), Daniela et al 2007). The assistance that stem cells provide may be due to their propensity to secrete useful trophic substances to limit the degree of cell death, down modulate the inflammation response to injury, and even provide a permissive substrate for neurons to grow upon (Ankey et al 2004, Silver and Miller 2004)). In addition, some adult stem cells may fuse with local cells or differentiate into a different cell type including neurons Figure 1. Cell types transplanted to human spinal cord 7-10 Ekim 2009, Antalya 199

3 OWENS J. Overview of Cellular Therapies in Spinal Cord Injuries and glia. However trans-differentiation probably does not contribute greatly to their restorative activity (Coutts and Keirstead 2007, Kim and Vellis 2009). In contrast to transplanted adult stem cells, embryonic stem cells can readily differentiate into any tissue type. That ability explains both ESCs potential value and as well as its potential danger. If ESCs are not directed down a specific lineage, as has been done to produce motor neurons and oligodendrocyte precursors, ESC can grow into teratomas or tumors within the CNS (Coutts ad Keirstead 2007). Although anecdotal reports from different countries around the world indicate that ESCs do promote recovery of function in SCI, there is a complete absence of published scientific literature on the activity of ESCs transplanted into the spinal cord in humans. However, a human trial with ESCs (directed to become oligodendrocyte precursors) has recently gained approval from the Federal Food and Drug Administration in the United States. Removing inhibition of axon regeneration There are two possible conceptual choices to eliminate inhibition of axon growth by the scar and from the myelin membrane (Fig. 2): The first is to eliminate the activity of each of inhibitors directly. Agents that can block myelin and scar inhibitors have been developed. The myelin-based inhibitors all converge (Li et al 2006) on a single receptor; the Nogo receptor (NogoR). This receptor can be blocked by a monoclonal antibody and also by a small peptide resembling the binding site on the Nogo inhibitor. The inhibitory moiety of the CSPGs can be removed by the bacterial enzyme Chondroitinase ABC. Both of these agents have been successful in eliminating axon growth inhibition in animal studies (Domeneiconi and Filbin 2005). The second option is to render the axon insensitive to the both classes of inhibitors by blocking the pathway that both scar and myelin inhibitors converge upon (Figure 2). The bacterial enzyme, C3 Transferase (Monnier et al 2006, Winston et al 2002) and surprisingly the over-the-counter drug, Ibuprophen (Fu et al 2007, Wang et al 2009) can block the activity of RhoA GTPase thereby uncoupling the activation of inhibitory receptors with axon growth and motility. Although the relief of inhibition may be better accomplished by using the drugs described above transplanted BMSCs can encourage cultured neurons to grow across inhibitory substrates (Opatz et al 2009, Wright et al 2007). OECs have been Figure 2. Inhibitors on myelin and scar Ulusal Hematoloji Kongresi

4 Overview of Cellular Therapies in Spinal Cord Injuries OWENS J. extensively explored as a solution to axon regeneration in the injured cord in both humans and animals (Richter, and Roskams 2007). In their natural environment OECs chaperone axons of the continuously replaced olfactory neurons through inhibitory territory within in the brain to their targets in the olfactory bulb. Unfortunately when transplanted into the damaged spinal cord their ability to assist axon growth over non-permissive substrates has not lived up to the hope that has been invested in these cells (Richter et al 2008). Restoration of dormant pathways Restoring function of dormant pathways appears to be the most successful aspect of cell transplantation. In animals, transplantation of BMSCs, Schwann cells, OECs and ESCs (converted to oligodendrocyte precursors) result in a substantial degree of remyelination, and thus restoring conduction in some pathways that were previously dormant (Ben-Hur and Goldman 2008). This activity is not a trivial contribution to functional recovery. Inducing axons to cross the scar cavity In the literature there is an abundance of data that demonstrate one of the most important impediments to healing after SCI is the formation of a complex scar at the site of the lesion. The scar is extensively decorated with CSPGs that inhibit the re-growth of axons after injury (Baritt et al 2006) ). There is some dispute about the need to surgically remove the scar or make the scar permeable to axons (Zhang et al 2007) during treatment for chronic SCI. This raises an obvious question: If the lesion scar is removed how should the cavity be filled to encourage axon growth? A biocompatible scaffold with horizontal linear axon guidance channels substantially increases the likelihood growing axons will transverse the scar region. In some studies seeding the scaffolding material with growth-promoting cells, including BMSCs or NSCs, improves the growth of axons across the lesion (Teng et al 2002). One group is working with human sympathetic ganglia neurons (Iwata et al 2006). During the culture of these cells the axons are aligned and stretched slowly until they reach a length of up to a centimeter. The entire array of neurons and their stretched axons can be transplanted into the spinal cord. This neural construct may provide an excellent bridge to transverse the lesion scar (Pfister et al 2006). Cell Replacement after SCI Although it may prove to be fairly easy to replace lost oligodendrocytes other lost cell types including lower motor neurons may prove to be more of a challenge. The loss of lower motor neurons due to SCI represents a major obstacle to overcome, however several investigators have managed convert stem cells from NSC, and ESCs to stable motor neuronal or glial phenotypes (Sharp and Keirstead 2007, De Filippis et al 2007) and have demonstrated in animal studies that some of these cells can integrate within the damaged spinal cord and function appropriately. Long term delivery of agents to the spinal cord A number of studies have conclusively confirmed that stem cells home to an injury site in CNS even when they have been introduced into the peripheral circulation (Akiyama et al 2002, Jendelova et al. 2004). The ability to target an injury in the CNS without invasive procedures in now being exploited by using BMSCs as a vehicle to deliver agents to an injury site within the CNS (Makar et al 2008, Nakar et al 2009). Regeneration of severed axons to their original synaptic targets may require prolonged (a year or more) and repetitive administration of regeneration-inducing agents. Using stem cells as delivery vehicles may be an ideal method for long term treatment of SCI. Future Directions Rejection of allogenic cells, even stem cells, is not a trivial concern in the development of therapy that involves transplantation to the CNS. There are conflicting reports regarding this issue, but it is fair to suggest that patient-specific donor cells should avoid rejection problems. A recent development in the science of stem cells is the creation induced pluripotent stem cells (ips). These cells derive from fully differentiated adult cells that have been re-programmed to form a stable converted cell type that possesses qualities of an embryonic stem cell, including pluripotency. This work has great potential for providing autologous cell types for SCI treatment (Amabile and Meissner 2009). Cells may provide a foundation for human SCI treatment, but to produce a substantial degree of recovery cells along with other therapeutic agents in a rational combination will be required to produce the degree of recovery people enduring paralysis so fervently desire (Lu et al 2004) Ekim 2009, Antalya 201

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