Stem Cells For The Treatment Of Spinal Cord Injury

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Experimental Neurology 209 (2008) 368 – 377 www.elsevier.com/locate/yexnr

Review

Stem cells for the treatment of spinal cord injury Margaret Coutts, Hans S. Keirstead ⁎ Reeve-Irvine Research Center, Stem Cell Research Center, Department of Anatomy and Neurobiology, 2111 Gillespie Neuroscience Research Facility, College of Medicine, University of California Irvine, Irvine, CA, USA Received 21 August 2007; revised 29 August 2007; accepted 1 September 2007 Available online 12 September 2007

Abstract This article reviews stem cell-based strategies for spinal cord injury repair, and practical issues concerning their translation to the clinic. Recent progress in the stem cell field includes clinically compliant culture conditions and directed differentiation of both embryonic stem cells and somatic stem cells. We provide a brief overview of the types of stem cells under evaluation, comparing their advantages and disadvantages for use in human clinical trials. We review the practical considerations and risks that must be addressed before human treatments can begin. With a growing understanding of these practical issues, stem cell biology, and spinal cord injury pathophysiology, stem cell-based therapies are moving closer to clinical application. © 2007 Elsevier Inc. All rights reserved. Keywords: Human embryonic stem cell; Neural stem cell; Bone marrow stem cell; Differentiation; Clinical trial; Regulatory

Introduction This review summarizes stem cell-based therapeutic strategies to treat spinal cord injury (SCI). Here we present the emerging perspective that stem cell administration is a viable therapeutic strategy and discuss the challenges to its development. Pre-clinical research has demonstrated the effectiveness of some stem cell-based treatments; however, several significant obstacles must be overcome before this research translates to the clinic. These hurdles include: identifying the best source of stem cells, optimizing their characteristics prior to transplant, reducing the risks of stem cell therapy, developing large-scale manufacturing technologies, and fulfilling regulatory considerations for government approval. Current treatments for SCI (Baptiste and Fehlings, 2007) include surgery to stabilize the injury site, high doses of corticosteroids to help limit secondary injury processes and rehabilitative care. Preclinical studies with neuroprotective agents (such as minocycline and the Rho antagonist, Cethrinr) are encouraging, and clinical trials with these agents will soon begin. While these treatment options may provide benefits, ⁎ Corresponding author. Fax: +1 949 824 5352. E-mail address: [email protected] (H.S. Keirstead). 0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2007.09.002

clinical improvements are modest and many patients still face significant neurologic dysfunction and disability. Stem cells offer several approaches for SCI repair. Stem cellbased therapies could, 1) replace damaged or diseased cells, 2) provide a cell-based electrical ‘relay’ between neurons above and below the injury, 3) ameliorate clinical deterioration and/or facilitate regeneration by providing neuroprotective or growth factors, or 4) play other indirect roles such as promoting neovascularization or providing a permissive substrate for regeneration of endogenous cells. Pathophysiology of SCI Transplanting stem cell derivates will not be sufficient to achieve complete functional restoration following SCI. The complex, reactive, and oftentimes multifocal nature of SCI presents several clinical challenges that must be overcome before stem cell-based therapies can become clinical realities. SCI results in inflammation, progressive hemorrhagic necrosis, edema, demyelination and cellular destruction. In the earlier stages of SCI, there is vascular destruction, a loss of neurons within the grey matter, and a loss of myelinating oligodendrocytes in the white matter. Axonopathy leads to a loss of functional connections (denervation) and retraction of the

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proximal axon. Cell death as a result of traumatic insult leads to further cell death via excitotoxicity. Excitotoxicity occurs as a result of the accumulation of excitatory molecules such as glutamate in the extracellular fluid, leading to overactivation of neurotransmitter receptors. High levels of calcium enter the cell and activate enzymes (phospholipases, proteases, etc.) that go on to damage cell structures. This cell damage and other changes often lead to induced cell death (apoptosis) and free radicalmediated lipid oxidation (Liu et al., 1997). Thus, during the first few days after injury there are many features detrimental to the survival and integration of transplanted cells (Hausmann, 2003). Inflammation plays a role in both the early and chronic stages of spinal cord injury (Hausmann, 2003; Fleming et al., 2006; Donnelly and Popovich, in press). Macrophages, neutrophils and T cells migrate in from the peripheral circulation and become activated. Microglia (normally resident in the spinal cord) also become activated. Both activated microglia and macrophages remove dead cells and debris via phagocytosis. The cytokines and chemokines that these cells produce propagate the inflammatory processes, as does complement activation (Anderson et al., 2004). Thus, inflammation in both the early and chronic stages of spinal cord injury could also damage transplanted cells. After the initial injury, the damage site expands from the injury epicenter (many centimeters in a human). Analysis of chronic SCI shows that, typically, portions of the more external white matter are spared while there is extensive damage of the more internally located grey matter. Within white matter, there is degeneration of both ascending and descending axons, and demyelination due to loss of oligodendrocytes. Chronic, progressive demyelination is a persistent feature of SCI (Totoiu and Keirstead, 2005). The astrocyte response begins immediately after injury (proliferation, hypertrophy, etc.) and evolves over time. Reactive astrocytes produce extracellular matrix components such as chondroitin and keratan sulfate proteoglycans. Ultimately, a scar-encapsulated cavity many times the size of the initial injury forms. The glial scar presents both a physical barrier and an inhibitory environment for axonal regeneration and remyelination (Fitch and Silver, 2008; Silver and Miller, 2004). Work from our laboratory strongly suggests that the gliotic environment of scarred, chronic lesions contributes to the failure of remyelination by prohibiting transplant-derived oligodendrocytes from remyelinating (Keirstead et al., 2005). Therefore, effective cell therapies for SCI likely necessitate transplantation during a brief therapeutic window: following acute inflammation, and prior to glial scar formation (Keirstead et al., 2005; Okano et al., 2003). Types of stem cells Stem cells can be divided into two broad categories, embryonic stem cells and somatic stem cells. Somatic stem cells include endogenous progenitor cells that repair and replace tissues in our bodies. The category of somatic stem cells also includes cells and cell lines derived from fetal tissues, neonatal tissues and adult tissues (e.g. neural stem cells, mesenchymal stem cells). These different types of stem cells will be briefly

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reviewed in this section, including a discussion of their potential for the treatment of SCI. Considerations include their potential for self-renewal, multipotency for providing a range of differentiated cell types, and the potential to meet clinical needs. Human embryonic stem cells Embryonic stem cells (ESCs) owe their discovery and potential to the early work of Kleinsmith and Pierce (1964). They demonstrated that a single embryonal carcinoma cell had the capacity to self-renew, and to generate multiple mature cell types. Later work with cell lines derived from the inner cell mass of cultured blastocyst stage embryos underscored the plasticity and potential of ESCs (Evans and Kaufman, 1981; Martin, 1981). A landmark experiment used ESCs to generate an entire mouse (Nagy et al., 1993). This astonishing plasticity was of interest not only to the scientific community, but also to medical community interested in clinical approaches to repair and replace damaged tissues. Successful isolation of human ESCs (hESCs) (Thomson et al., 1998) further heightened the interest in the field. ESCs can be prepared from pre-implantation or blastocyst-stage embryos (from unused embryos created during in vitro fertilization procedures), by somatic cell nuclear transfer, or by parthenogenetic activation of eggs (Cibelli et al., 2002; Vrana et al., 2003). Unlike normal somatic cells, ESCs can be grown in virtually unlimited quantities because they do not undergo senescence, retain high telomerase activity and normal cell cycle signaling. With proper culture techniques, they do not undergo the genomic, mitochondrial and epigenetic changes that lead to transformation (Zeng and Rao, 2007). Of all stem cell types, ESCs currently show the greatest potential for the widest range of cell replacement therapies. They can be propagated in vitro almost indefinitely, can be stably banked, and maintain a normal karyotype and differentiation potential even after years of culture. These attributes make hESCs a commercially viable possibility for production-scale processes. Originally, hESCs where cultivated on mouse fibroblast feeder layers with medium containing bovine serum. Great progress has been made in cultivating these cells, as current approaches use defined media with few or no reagents derived from animal sources (Richards et al., 2002; Lee et al., 2005). This is important to preclude the possibility of cellular incorporation of zoonotic pathogens from the media. Other types of stem cells are not as thoroughly characterized, and the methods for directing hESC differentiation are most advanced. Several studies have indicated that rodent ESCs can be directed in their differentiation to neuronal (Finley et al., 1996; Lang et al., 2004) or glial fates (Liu et al., 2000; Billon et al., 2006). ESC-derived neurons can survive, integrate and help restore function following transplantation into spinal cord injured rats (Deshpande et al., 2006). Human ESCs have been directed to differentiate into multipotent neural precursors (Carpenter et al., 2001; Reubinoff et al., 2001), into low-purity motor neurons (Li et al., 2005) and recently into highpurity oligodendrocyte progenitors (Keirstead et al., 2005; Nistor et al., 2005). Cumulatively, these studies demonstrate that the

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directed differentiation of hESCs into high-purity neural populations is possible. Neural stem cells Endogenous neural stem cells (NSCs) exist within the central nervous system (CNS) of higher mammals, and recently, several groups have successfully isolated and expanded human NSCs from specific regions of the developing and adult brain (Snyder et al., 1992; Lois and Alvarez-Buylla, 1993; Uchida et al., 2000) (Reynolds and Weiss, 1992), spinal cord (Mayer-Proschel et al., 1997) and optic nerve (Shi et al., 1998). NSCs are of particular interest for SCI repair. It is believed that they are already committed to a neural fate and hence will be easier to differentiate into mature neural phenotypes, and less likely than ESCs to become neoplastic. NSCs are neurogenic and gliogenic within the CNS throughout both development and adulthood. They can be expanded in vitro by exposure to different growth factors, maintain some capacity for self-renewal even after several freeze–thaw cycles, and are capable of generating differentiated progeny that can functionally integrate (Caldwell et al., 2001) and repair the damaged CNS (Nunes et al., 2003; Cummings et al., 2006; Ogawa et al., 2002; Iwanami et al., 2005). NSCs can also protect against excitotoxicity and secrete neurotrophic growth factors (Llado et al., 2004; Lu et al., 2003). Multipotent NSCs have recently been isolated from adult human subcortical white matter, and can be maintained in vitro before being transplanted into fetal rat brain, where they generate functionally competent neurons and glia (Nunes et al., 2003). However, adult NSCs divide less frequently than their embryonic counterparts and therefore may be more difficult to expand into large cultures required for clinical applications (Doetsch et al., 1999; Morshead et al., 1998). Besides their limited replication potential, there is evidence that the differentiation potential of NSCs decreases with time in culture (Wright et al., 2006). In addition, differentiation of NSCs into high purity populations has not been demonstrated, although progress has been made to increase the percentage of either neurons or astrocytes during in vitro differentiation using different combinations of growth factors (Caldwell et al., 2001; Han et al., 2002) or alternate growth conditions (Yan et al., 2007). Nonetheless, transplanted multipotent NSCs have been shown to reach regions of tissue damage, differentiate into myelinating oligodendrocytes, and cause clinical improvement following intraventricular, intravenous, intraspinal, or intraperitoneal delivery to various demyelinating or dysmyelinating animal models (Einstein et al., 2003; Pluchino et al., 2003) (Ben-Hur et al., 2003; Bulte et al., 2003). Approximately 35–40 reports have described neural stem cell treatments for SCI (reviewed in Enzmann et al., 2006); most have used brain-derived NSC. Many of these studies showed that transplanted NSCs are able to generate astrocytes and oligodendrocytes very effectively. Generally, the production of neurons was low or not detectable. It has been suggested that the adult SCI environment is conducive for the differentiation of NSCs to oligodendrocytes or astrocytes, but for undefined

reasons, it is not as conducive for neuronal differentiation. Some studies show that more mature populations of neural precursors will generate neurons, either because these cells do not respond to inhibitory signals, or alternatively, no longer need positive environmental influences to attain a neuronal phenotype (Han et al., 2002; Roy et al., 2004; Yan et al., 2007). Human neural tissue is generally obtained from cadavers, which restricts supply. Furthermore, human neural tissue has a limited expansion potential in vitro. These factors limit the usefulness of NSCs in the clinical setting. Olfactory ensheathing cells Olfactory ensheathing cells (OECs) are support cells that wrap olfactory axons and facilitate their regeneration throughout the life of mammalian species. They are reported to have exceptional plasticity, and importantly, allow neurons to cross a glial scar as well as the PNS–CNS boundary (reviewed in (Richter and Roskams, 2008) and (Raisman and Li, 2007)). These cells are relatively easy to obtain from nasal biopsies and could provide a source of autologous cells for transplant. Over the last decade, a number of labs have used OECs in several different acute and chronic models of rodent SCI. In some cases, remyelination of axons and regeneration of damaged axons was reported along with a surprising degree of functional recovery. Other groups have not been able to reproduce these results, due perhaps in part to differences in biological properties of primary OECs with increasing age and/or passage number (Pastrana et al., 2006). While there is great contention in the field, the majority of reports hold that these cells are supportive in repair processes, but the evidence that they facilitate regeneration of long axonal tracts is limited. In addition, it is not yet clear whether they can be expanded in sufficient numbers for use in human cell replacement strategies. Mesenchymal stem cells Mesenchymal stem cells (MSC), bone marrow mononuclear cells and umbilical cord blood are potentially rich sources of stem cells and a number of studies have used them to treat CNS damage. Some of these studies have shown promising results, however, basic knowledge concerning their mechanism of action and therapeutic potential is lacking. Despite the lack of understanding of the underling mechanisms, clinical trials for SCI treatments are beginning (Yoon et al., 2007; Callera and do Nascimento, 2006). MSC and bone marrow-derived cells have epitomized a central question in stem cell biology: whether stem cells from one tissue can generate cells of another. The discovery of Ychromosome-labeled neurons (Mezey et al., 2003), Purkinje cells (Weimann et al., 2003) or hippocampal cells (Cogle et al., 2004) within the brains of women who had received bone marrow transplants from men ignited controversy of whether this was a rare fusion event, or evidence of stem cell plasticity that could lead to useful therapies. The application of these types of stem cells for CNS repair has been reviewed recently (Parr et al., 2007; Enzmann et al.,

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2006). These reviews take on the difficult task of comparing studies with greatly heterogeneous starting materials and differences in injury models, immunosuppression regimes, methods of transplantation, etc. The effort to evaluate these studies is well warranted, given the practical advantages of MSC and bone marrow-derived cells: they are easily obtainable, autologous transplantation is possible, they may be immuno-privileged, and they have the ability to migrate to areas of damage and inflammation. Most of these studies report improved function as a result of implantation (usually determined by a subjectivelyscored locomotor test, though some use field potential recording (Akiyama et al., 2002a,b), and differentiation to oligodendrocytes, or less frequently neurons, weeks to months after transplantation. Some of the most convincing studies demonstrated that LacZ or GFP pre-labeled cells localized to Schwann- and oligodendrocyte-like cells following transplantation (Akiyama et al., 2002a,b) (Akiyama et al., 2002a,b; Sasaki et al., 2001). Opposite results were obtained in other studies: there was no detection of transdifferentiation in the transplanted cells, even though functional improvement was noted (Koda et al., 2005). How can these disparate results be reconciled? A broad survey of the literature indicates that differentiation of transplanted MSC and bone marrow stem cells is dictated by the environment, a concern that is made more relevant by the fact that means of in vivo differentiation to high-purity neural populations are lacking. Some degree of differentiation may well be possible, especially given that a subset of ex vivo bone marrow-derived cells express neuronal and oligodendroglial markers (Goolsby et al., 2003; Steidl et al., 2002). Besides directly replacing damaged oligodendrocytes and neurons, bone marrow cells and MSC could play an important supportive role in SCI therapies. They could create a more favorable environment for limiting damage and promoting regeneration, via immunoregulation (Aggarwal and Pittenger, 2005; Noel et al., 2007), expression of growth factors and cytokines (Song et al., 2004), improved vascularization, providing a permissive growth substrate, and/or suppressing cavity formation (Hofstetter et al., 2002). These different mechanisms are not mutually exclusive and a number of them could contribute to improved outcomes. Indeed, naive and genetically-modified MSC have been used in combinatorial therapies in animal models of SCI (Lu et al., 2005; Lu et al., 2004).

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In contrast to the lack of neuronal regeneration within the adult CNS, glial regeneration is successful following some insults to the CNS. Endogenous oligodendrocyte precursors can proliferate and differentiate in response to various types of injuries (Keirstead et al., 1998; McTigue et al., 2001; Wolswijk, 2000). Cell division is a prerequisite for remyelination (Keirstead et al., 1998), which is characterized by thin and short myelin sheaths (Totoiu and Keirstead, 2005) (Prineas et al., 1993). Interestingly, remyelination is dependent on the re-expression of developmentallyregulated genes (Capello et al., 1997), though it differs in some respects from transcription in development (Ibanez et al., 2003). While oligodendrocytes proliferate and differentiate in response to SCI, there is a net loss of myelin. Demyelination as a chronic, progressive problem in SCI (Guest et al., 2005; Totoiu and Keirstead, 2005). Remyelination is less efficient in old animals than in young animals (Shields et al., 2000) and less efficient after repeated episodes of demyelination (Mason et al., 2004). It is possible that depletion of myelinogenic progenitors contributes to remyelination failure. Mature oligodendrocytes are incapable of remyelinating axons (Keirstead and Blakemore, 1997; Crang et al., 1998) and there is no convincing evidence that differentiated oligodendrocytes are able to revert to a progenitor state. In addition, astrogliosis could contribute to the failure of remyelination, forming a physical barrier and blocking access of oligodendrocyte progenitors to demyelinated axons (Keirstead et al., 2005; Ibanez et al., 2003). Astrocytes can also express inhibitory molecules such as Jagged1 (which inhibits oligodendrocyte differentiation and process outgrowth) (John et al., 2002). Failure of remyelination is probably due to a combination of environmental factors and innate characteristics of endogenous oligodendrocyte progenitors. In addition to directly replacing damaged neurons and oligodendrocytes, stem cell therapies could also play an indirect role by supporting endogenous stem cells. Transplanted cells could provide trophic factors (Zhang et al., 2006) or serve as a substrate permissive for growth, differentiation, elongation or connection to other cells. Because the glial scar takes weeks to form a thick, rubbery obstruction, there is likely a ‘window of opportunity’ following injury during which the impediments to endogenous or transplant-mediated regeneration are fewer. This period of time presents an opportunity for stem cell-derived therapies to repair SCI.

Endogenous stem cells and progenitors Clinical and scientific challenges Neural stem cells (NSC) are present in the adult spinal cord, however, it is clear that the capacity of endogenous NSCs to replace lost cells after SCI is poor. Axonal regeneration from pre-existing neurons is also poor, and it is likely that many of the same factors that prevent axonal regeneration also inhibit the function of endogenous NSC, neural progenitors and mature neurons. These factors include the formation of the glial scar, the lack of neurotrophic factors, inhibitory sulfated proteoglycans, and inhibitory myelin-associated molecules (reviewed in (Ramer et al., 2005) and (Fitch and Silver, 2008)). Falling cAMP levels may also be inhibitory to regenerating cells (Pearse et al., 2004) and differentiating progenitors.

Potential stem cell therapies have a number of clinical and technical issues that need to be resolved before human clinical trials can begin. It will not be necessary to answer all of the questions regarding underlying molecular mechanisms of action, but there must be clear evidence that stem cell therapies are safe, provide improved function and that the benefits outweigh the risks. Most important is an understanding of the potential dangers of stem cell-based therapy. Preclinical studies will be essential in answering these questions; however, there will always be some uncertainties when translating preclinical animal studies to the human condition. Hence, collective ‘buy-

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in’ from the scientific and medical communities is a critical prerequisite to a clinical trial. Do no harm A number potential adverse effects must be evaluated before a stem cell-based therapeutic can be applied to humans. The risks include tumorigenesis, immunological complications, allodynia (pain), or complications associated with an unexpected change in phenotype of the transplanted cells (for example, dedifferentiation or excess proliferation). Tumor formation is a significant concern for transplant strategies involving embryonic stem cells; however, this risk decreases as the cells become more highly differentiated (i.e. less multipotent). Transplanted ESCs by definition form teratomas, which consist of endodermal, mesodermal and ectodermal lineages (Reubinoff et al., 2001). However, no rational medical researcher would consider using undifferentiated ESCs in a human therapeutic strategy. Somatic stem cells are less proliferative and less multipotent; hence they are considered less tumorigenic. Another important concern is immune rejection of the transplant. The host's immune system could destroy the implanted cells and thereby eliminate any benefit that they conferred. Furthermore, the inflammatory response associated with cell rejection could cause additional harm to the patient. HLA matching of the stem cell transplant to the host is one method to avoid immune rejection. Unfortunately, the number of available stem cell lines is limited, and it is likely that life-long immunosuppression will be necessary to prevent rejection of the transplanted cells. The side-effects and risks of immunosuppression are significant and include nausea, vomiting, diarrhea, liver and kidney toxicity, lowered counts of leukocytes and platelets, and increased susceptibility to infections and malignancies (Habwe, 2006). Also, it is not clear what effect immunosuppression will have on the transplanted cells. This is not a trivial issue, as evidenced by the fact that pancreatic islet transplantation was not successful until immunosuppression regimes were developed that were less toxic to the transplanted cells (Nanji and Shapiro, 2004). It is intriguing that ESC and ESC-derived cells may not generate the same degree of immune response associated with other transplanted tissues (Drukker et al., 2006; Utermohlen and Kronke, 2007). Such studies should be repeated with the differentiated cells of interest. It is conceivable that transplant rejection may be overcome by tolerizing recipients prior to transplantation (Salama et al., 2001; Rosengard and Turka, 2001). Other potential means of avoiding transplant rejection include generating stem cells that are immunologically compatible with recipients using somatic cell nuclear transfer (SCNT—deriving a stem cell line by transferring nuclei of recipient into donor stem cell or egg) or by developing a “universal donor” ESC line (Lengerke et al., 2007). It remains to be seen if these later approaches will be successful or commercially viable. Allodynia is a significant concern for stem cell-based strategies to treat SCI. In a thorough and thoughtful investigation, Hoffstetter and colleagues recently underscored the ben-

efits and dangers of cell-based therapies for SCI (Hofstetter et al., 2005). In these studies, NSCs were transplanted into the low-thoracic spinal cord of rats 1 week after injury. Functional recovery was noted in the affected hind limbs, but abnormal, painful sensitivity developed in the forepaws (which had been unaffected by the injury). Histology indicated that the transplant had differentiated in situ to a predominantly astrocytic phenotype, and that these astrocytes promoted sprouting of sensory fibers within the spinal cord that were associated with allodynia. When the NSCs were directed to produce oligodendrocytes, further functional gains were attained in the hind limbs and allodynia was avoided. These results have been verified by other workers (Hendricks et al., 2006). Careful studies such as these are critical to the preclinical development of stem cell-based therapies. Nonetheless, it should be appreciated that unexpected adverse effects do occur in human clinical trials, despite exhaustive preclinical development. Differentiation to undesirable cell types is a risk inherent to all multipotent cells. The NSCs used by Hoffstetter and colleagues had the potential to produce neurons or oligodendrocytes in vitro, but in the complex environment of the injured spinal cord they produced mostly astrocytes (Hofstetter et al., 2005). This underscores the importance of evaluating the phenotype of the cells after transplant. In addition to evaluating phenotype, preclinical studies must also evaluate proliferation and migration of the transplanted cells. Uncontrolled proliferation and migration is obviously undesirable; spreading beyond the implant site would increase the risk of adverse events such as cerebrospinal fluid occlusion or emboli causing stroke. The nature of the transplant Preclinical studies will guide decisions on the best transplant position within the spinal cord, the number of implantation sites, the appropriate cell number to transplant, and the optimal timing of transplants relative to the onset of injury. The optimal time for transplantation of cells into the injured spinal cord is likely after acute inflammation and excitotoxicity has subsided, and prior to the formation of extensive glial scar. Other practical matters concerning the transplant include efficient and safe methods to expand and differentiate stem cells. It is imperative that the cells maintain genetic and epigenetic stability, and do not become senescent. The methods to induce differentiation must also be efficient, so a high percentage of cells attain the desired phenotype. A central question concerning the transplant is the optimal degree of differentiation. While a less differentiated cell may better respond to environmental cues and show enhanced capacity for migration and growth, a more differentiated cell may mitigate the risks of inappropriate differentiation and neoplasia. A broad review of the literature clearly indicates that lineage restriction to the progenitor stage is essential to limit tumor formation and differentiation of undesirable cell types, and enhance integration. Lineage restriction prior to transplantation overcomes limitations of the environment, which may not provide appropriate cues for targeted differentiation (for example, efficient generation of neurons is limited in non-

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neurogenic regions of the CNS (Song et al., 2002; Shihabuddin et al., 2000)). The directed differentiation of stem cells to high purity lineages has recently been achieved (Nistor et al., 2005; Keirstead et al., 2005). This is a critical advance in stem cell biology since it allows researchers to generate a potentially inexhaustible supply of relatively pure, committed, lineagespecific cells for transplantation. Stem cells are particularly amenable to genetic modification, offering the potential to combine cell replacement therapy with small molecule approaches to repair. Modifying the transplant population to secrete growth factors or functional blockers of endogenous inhibitors may augment the effect of cell replacement on injury pathogenesis or repair. The use of a reporter genes permit sorting of differentiated cells from residual undifferentiated ES cells, mitigating the risk of tumor formation (Tang et al., 2002). However, there are inherently unpredictable consequences when modifying the genome, and regulatory agencies are understandably cautious with these methods. A poignant example of this risk was realized in a gene therapy trial to treat severe combined immunodeficiency, when the replacement gene and its vector integrated into the patient genome near a proto-oncogene, resulting in leukemia (Buckley, 2002).

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combined cell transplantation with minocycline treatment and growth factor (PDGF-AA, bFGF and EGF) delivery to enhance cell survival (Karimi-Abdolrezaee et al., 2006). Findings from the laboratory of Aileen Anderson indicated that multipotent human CNS stem cells derived from fetal brain could be maintained as neurospheres, and that their transplantation into sites of moderate spinal cord contusion in adult NOD-scid mice resulted in differentiation to neurons and oligodendrocytes, remyelination by transplanted cells and improved locomotion (Cummings et al., 2005, 2006). Findings from the laboratory of Lars Olson indicated that transduction of neural stem cells isolated from adult rat spinal cords with neurogenin 2 depressed astroglial differentiation and increased oligodendroglial differentiation, and that their transplantation into sites of weight-drop injury in adult rats resulted in remyelination and improved locomotion; notably, in the absence of neurogenin 2 transduction, neural stem cells differentiated primarily into astrocytes after transplantation, and caused aberrant axonal sprouting associated with allodynia-like hypersensitivity of the forepaws (Hofstetter et al., 2005). These studies clearly indicate that myelinogenic transplants elicit functional recovery following SCI. However, in all instances the mechanism of action is unknown.

Evaluating success Regulatory concerns Ideally, stem cell-based therapies should be assessed using cellular, physiological and behavioral assays. Many published studies document functional improvements, but do not characterize the cells post-transplant nor demonstrate how the cells contributed to the outcome. Conversely, some publications provide evidence of successful differentiation using molecular and immunological methods, but do not report behavioral outcome. Safety studies are rarely undertaken by the scientific community, which prioritizes discovery over development. While this work is scientifically significant, it will be important to have a more complete picture before human clinical trials can begin. Ideally, promising findings should be replicated in independent laboratories prior to the initiation of clinical trials. A recent concurrence of data from multiple laboratories has led to the conclusion that myelinogenic transplants elicit histologic repair and functional recovery following SCI. Findings from our laboratory indicated that hESCs can be manipulated in vitro to generate high purity human oligodendrocyte progenitor cells (OPCs) in large quantities (Nistor et al., 2005), and that their transplantation into sites of moderate spinal cord contusion in adult rats results in differentiation to mature oligodendrocytes, remyelination by transplanted cells and improved locomotion (Keirstead et al., 2005). Follow-up studies indicated that the procedure was safe, in that the transplant was not associated with tumor formation, scarring, tissue pathogenesis, or behavioral decline (Cloutier et al., 2006). Findings from the laboratory of Michael Fehlings indicated that multipotent adult neural precursor cells can be isolated from the subventricular zone of the forebrain, and that their transplantation into sites of aneurysm clip-induced spinal cord compression in adult rats resulted in differentiation to mature oligodendrocytes, remyelination by transplanted cells and improved locomotion; this study

Regulatory bodies such as the Food and Drug Administration (FDA) and Institutional Review Boards (IRBs) will guide the translation of stem cell therapies for SCI in animals to human clinical trials. Researchers, biotechnology companies, patients and patient advocates should be aware of the regulatory issues that must be addressed before human trials can begin. These new experimental therapies will have special concerns. First, the spinal cord is a particularly sensitive and risky site to treat. Secondly, stem cell therapy is novel and entails a number of complex issues that have never been addressed before. The regulatory issues span categories as diverse as pharmacology and toxicology, rules concerning the manufacture of cell and tissue based products, xeno- and allogenenic transplantation, and surgical methods. At a minimum, a stem cell-based transplant must be well defined in terms of sterility, purity, potency, identity, stability, safety and efficacy. One regulatory issue common to many different types of clinical trials is the concept of “process control”. The stem cells and all of the materials that come in contact with them throughout their preparation must have a completely traceable identity and source. Further, the reagents must have documented quality assurances and quality controls. The stem cells themselves must be screened for the presence of retroviruses and infectious pathogens. Karyotypic analysis must be performed to ensure there are no gross genetic changes. There can be no undifferentiated embryonic stem cells in the transplant population, which may have the capacity to form tumors. This last qualification can be accomplished by checking for predetermined markers such as OCT4, SSEA4, and TRA81. Other critical considerations include four toxicological parameters: migration of the cell transplant, inappropriate

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cellular differentiation, the possibility of tumor formation, and host immune responses. Cell migration (bio-distribution) from the implant site can be evaluated in preclinical testing, using reverse transcription polymerase chain reaction (RT-PCR) for specific markers on isolated organs following transplantation. Inappropriate cellular differentiation must also be defined in preclinical testing. Clinical deficits could be worsened if, for example, stem cells transplanted into the spinal cord of a patient inappropriately differentiated into cartilage-producing cells. Inappropriate differentiation could also include de-differentiation to a pluripotent state, which would be a risk for tumor formation. Tumor formation is of particular concern because the cells implanted in the spinal cord would be difficult or impossible to remove. Host immune responses must be evaluated in terms of potential harm both to the host and to the implant. Since the cells are foreign to the body, some immune response is expected. Immunosuppression will probably be required, as will histologic studies to define any immune activation. Before clinical trials can begin, standard operating procedures must be compiled to standardize the methods used in preparing and delivering the cell therapy (including cell cultivation and differentiation methods, transplantation surgery, etc.). Surgical teams will need to be assembled and well trained in every aspect of the transplantation. Equipment may be subject to additional regulatory requirements prior to conducting human trials. While much of the equipment is used routinely for clinical neurosurgical practices (e.g. syringes, infusion pumps, stereotaxic equipment), the application to stem cell clinical trials will require additional study. Preclinical studies using animals have developed standard operating procedures, however some aspects must be translated to human scale, such as the maximum volume and number of cells that can be transplanted, or the number of injection sites. Conclusions and summary The literature over the past few decades clearly documents a growing appreciation of the very complicated pathophysiology of SCI. During this time, the field of stem cell biology has emerged. This new field has the potential to bring therapies to previously untreatable diseases and injuries. Animal models have shown some positive results, validating scientific principles and strategies. In addition, progress has been made with a number of practical concerns. Commercial-scale production of NSC and ESC cultures is progressing, a necessary step in creating a sufficient volume of cells for human trials. Great improvements have been made towards creating “defined” growth conditions to yield safe, transplantable cells, free from human and zoonotic pathogens. Differentiation protocols are improving, yielding cells of higher purity and better function. Spinal cord researchers are appreciating the complexity of SCI, and appreciate that a multifaceted approach will be needed (for example, differentiated stem cells, plus trophic factors and tissue engineering of a permissive environment). These experiments will be difficult to design and execute such that meaningful results can be obtained. Unquestionably, there must be an accumulation of studies that show functional recovery in animal

models before risking treatments in human patients. Patients, clinicians, scientists and regulatory agencies must also consider longer-term safety issues of stem cell therapies, including the likelihood of life-long immunosuppressive regimes. While attaining a cure will be difficult, the spinal cord research field should acknowledge the complexity of the challenge and not be deterred from pursuing it. References Aggarwal, S., Pittenger, M.F., 2005. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105, 1815–1822. Akiyama, Y., Radtke, C., Honmou, O., Kocsis, J.D., 2002a. Remyelination of the spinal cord following intravenous delivery of bone marrow cells. Glia 39, 229–236. Akiyama, Y., Radtke, C., Kocsis, J.D., 2002b. Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J. Neurosci. 22, 6623–6630. Anderson, A.J., Robert, S., Huang, W., Young, W., Cotman, C.W., 2004. Activation of complement pathways after contusion-induced spinal cord injury. J. Neurotrauma 21, 1831–1846. Baptiste, D.C., Fehlings, M.G., 2007. Update on the treatment of spinal cord injury. Prog. Brain Res. 161, 217–233. Ben-Hur, T., Einstein, O., Mizrachi-Kol, R., Ben-Menachem, O., Reinhartz, E., Karussis, D., Abramsky, O., 2003. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia 41, 73–80. Billon, N., Jolicoeur, C., Raff, M., 2006. Generation and characterization of oligodendrocytes from lineage-selectable embryonic stem cells in vitro. Methods Mol. Biol. 330, 15–32. Buckley, R.H., 2002. Gene therapy for SCID—a complication after remarkable progress. Lancet 360, 1185–1186. Bulte, J.W., Ben-Hur, T., Miller, B.R., Mizrachi-Kol, R., Einstein, O., Reinhartz, E., Zywicke, H.A., Douglas, T., Frank, J.A., 2003. MR microscopy of magnetically labeled neurospheres transplanted into the Lewis EAE rat brain. Magn. Reson. Med. 50, 201–205. Caldwell, M.A., He, X., Wilkie, N., Pollack, S., Marshall, G., Wafford, K.A., Svendsen, C.N., 2001. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat. Biotechnol. 19, 475–479. Callera, F., do Nascimento, R.X., 2006. Delivery of autologous bone marrow precursor cells into the spinal cord via lumbar puncture technique in patients with spinal cord injury: a preliminary safety study. Exp. Hematol. 34, 130–131. Capello, E., Voskuhl, R.R., McFarland, H.F., Raine, C.S., 1997. Multiple sclerosis: re-expression of a developmental gene in chronic lesions correlates with remyelination. Ann. Neurol. 41, 797–805. Carpenter, M.K., Inokuma, M.S., Denham, J., Mujtaba, T., Chiu, C.P., Rao, M.S., 2001. Enrichment of neurons and neural precursors from human embryonic stem cells. Exp. Neurol. 172, 383–397. Cibelli, J.B., Grant, K.A., Chapman, K.B., Cunniff, K., Worst, T., Green, H.L., Walker, S.J., Gutin, P.H., Vilner, L., Tabar, V., Dominko, T., Kane, J., Wettstein, P.J., Lanza, R.P., Studer, L., Vrana, K.E., West, M.D., 2002. Parthenogenetic stem cells in nonhuman primates. Science 295, 819. Cloutier, F., Siegenthaler, M.M., Nistor, G., Keirstead, H.S., 2006. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm. Regen. Med. 1, 469–479. Cogle, C.R., Yachnis, A.T., Laywell, E.D., Zander, D.S., Wingard, J.R., Steindler, D.A., Scott, E.W., 2004. Bone marrow transdifferentiation in brain after transplantation: a retrospective study. Lancet 363, 1432–1437. Crang, A.J., Gilson, J., Blakemore, W.F., 1998. The demonstration by transplantation of the very restricted remyelinating potential of post-mitotic oligodendrocytes. J. Neurocytol. 27, 541–553. Cummings, B.J., Uchida, N., Tamaki, S.J., Salazar, D.L., Hooshmand, M., Summers, R., Gage, F.H., Anderson, A.J., 2005. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc. Natl. Acad. Sci. U. S. A. 102, 14069–14074.

M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377 Cummings, B.J., Uchida, N., Tamaki, S.J., Anderson, A.J., 2006. Human neural stem cell differentiation following transplantation into spinal cord injured mice: association with recovery of locomotor function. Neurol. Res. 28, 474–481. Deshpande, D.M., Kim, Y.S., Martinez, T., Carmen, J., Dike, S., Shats, I., Rubin, L.L., Drummond, J., Krishnan, C., Hoke, A., Maragakis, N., Shefner, J., Rothstein, J.D., Kerr, D.A., 2006. Recovery from paralysis in adult rats using embryonic stem cells. Ann. Neurol. 60, 32–44. Doetsch, F., Caille, I., Lim, D.A., Garcia-Verdugo, J.M., Alvarez-Buylla, A., 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716. Donnelly, D.J., Popovich, P.G., 2008. Inflammation and its role in neuroprotection, axonal regeneration and functional recovery after spinal cord injury. Exp. Neurol. 209, 378–388. Drukker, M., Katchman, H., Katz, G., Even-Tov Friedman, S., Shezen, E., Hornstein, E., Mandelboim, O., Reisner, Y., Benvenisty, N., 2006. Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 24, 221–229. Einstein, O., Karussis, D., Grigoriadis, N., Mizrachi-Kol, R., Reinhartz, E., Abramsky, O., Ben-Hur, T., 2003. Intraventricular transplantation of neural precursor cell spheres attenuates acute experimental allergic encephalomyelitis. Mol. Cell. Neurosci. 24, 1074–1082. Enzmann, G.U., Benton, R.L., Talbott, J.F., Cao, Q., Whittemore, S.R., 2006. Functional considerations of stem cell transplantation therapy for spinal cord repair. J. Neurotrauma 23, 479–495. Evans, M.J., Kaufman, M.H., 1981. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156. Finley, M.F., Kulkarni, N., Huettner, J.E., 1996. Synapse formation and establishment of neuronal polarity by P19 embryonic carcinoma cells and embryonic stem cells. J. Neurosci. 16, 1056–1065. Fitch, M.T., Silver, J., 2008. CNS injury, glial scars, and inflammation: inhibitory extracellular matrices and regeneration failure. Exp. Neurol. 209, 294–301. Fleming, J.C., Norenberg, M.D., Ramsay, D.A., Dekaban, G.A., Marcillo, A.E., Saenz, A.D., Pasquale-Styles, M., Dietrich, W.D., Weaver, L.C., 2006. The cellular inflammatory response in human spinal cords after injury. Brain 129, 3249–3269. Goolsby, J., Marty, M.C., Heletz, D., Chiappelli, J., Tashko, G., Yarnell, D., Fishman, P.S., Dhib-Jalbut, S., Bever Jr., C.T., Pessac, B., Trisler, D., 2003. Hematopoietic progenitors express neural genes. Proc. Natl. Acad. Sci. U. S. A. 100, 14926–14931. Guest, J.D., Hiester, E.D., Bunge, R.P., 2005. Demyelination and Schwann cell responses adjacent to injury epicenter cavities following chronic human spinal cord injury. Exp. Neurol. 192, 384–393. Habwe, V.Q., 2006. Posttransplantation quality of life: more than graft function. Am. J. Kidney Dis. 47, S98–S110. Han, S.S., Kang, D.Y., Mujtaba, T., Rao, M.S., Fischer, I., 2002. Grafted lineage-restricted precursors differentiate exclusively into neurons in the adult spinal cord. Exp. Neurol. 177, 360–375. Hausmann, O.N., 2003. Post-traumatic inflammation following spinal cord injury. Spinal Cord 41, 369–378. Hendricks, W.A., Pak, E.S., Owensby, J.P., Menta, K.J., Glazova, M., Moretto, J., Hollis, S., Brewer, K.L., Murashov, A.K., 2006. Predifferentiated embryonic stem cells prevent chronic pain behaviors and restore sensory function following spinal cord injury in mice. Mol. Med. 12, 34–46. Hofstetter, C.P., Holmstrom, N.A., Lilja, J.A., Schweinhardt, P., Hao, J., Spenger, C., Wiesenfeld-Hallin, Z., Kurpad, S.N., Frisen, J., Olson, L., 2005. Allodynia limits the usefulness of intraspinal neural stem cell grafts; directed differentiation improves outcome. Nat. Neurosci. 8, 346–353. Hofstetter, C.P., Schwarz, E.J., Hess, D., Widenfalk, J., El Manira, A., Prockop, D.J., Olson, L., 2002. Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc. Natl. Acad. Sci. U. S. A. 99, 2199–2204. Ibanez, C., Shields, S.A., El-Etr, M., Leonelli, E., Magnaghi, V., Li, W.W., Sim, F.J., Baulieu, E.E., Melcangi, R.C., Schumacher, M., Franklin, R.J., 2003. Steroids and the reversal of age-associated changes in myelination and remyelination. Prog. Neurobiol. 71, 49–56. Iwanami, A., Kaneko, S., Nakamura, M., Kanemura, Y., Mori, H., Kobayashi, S., Yamasaki, M., Momoshima, S., Ishii, H., Ando, K., Tanioka, Y., Tamaoki, N.,

375

Nomura, T., Toyama, Y., Okano, H., 2005. Transplantation of human neural stem cells for spinal cord injury in primates. J. Neurosci. Res. 80, 182–190. John, G.R., Shankar, S.L., Shafit-Zagardo, B., Massimi, A., Lee, S.C., Raine, C.S., Brosnan, C.F., 2002. Multiple sclerosis: re-expression of a developmental pathway that restricts oligodendrocyte maturation. Nat. Med. 8, 1115–1121. Karimi-Abdolrezaee, S., Eftekharpour, E., Wang, J., Morshead, C.M., Fehlings, M.G., 2006. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J. Neurosci. 26, 3377–3389. Keirstead, H.S., Blakemore, W.F., 1997. Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord. J. Neuropathol. Exp. Neurol. 56, 1191–1201. Keirstead, H.S., Levine, J.M., Blakemore, W.F., 1998. Response of the oligodendrocyte progenitor cell population (defined by NG2 labelling) to demyelination of the adult spinal cord. Glia 22, 161–170. Keirstead, H.S., Nistor, G., Bernal, G., Totoiu, M., Cloutier, F., Sharp, K., Steward, O., 2005. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25, 4694–4705. Kleinsmith, L.J., Pierce Jr., G.B., 1964. Multipotentiality of single embryonal carcinoma cells. Cancer Res. 24, 1544–1551. Koda, M., Okada, S., Nakayama, T., Koshizuka, S., Kamada, T., Nishio, Y., Someya, Y., Yoshinaga, K., Okawa, A., Moriya, H., Yamazaki, M., 2005. Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport 16, 1763–1767. Lang, K.J., Rathjen, J., Vassilieva, S., Rathjen, P.D., 2004. Differentiation of embryonic stem cells to a neural fate: a route to re-building the nervous system? J. Neurosci. Res. 76, 184–192. Lee, J.B., Lee, J.E., Park, J.H., Kim, S.J., Kim, M.K., Roh, S.I., Yoon, H.S., 2005. Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition. Biol. Reprod. 72, 42–49. Lengerke, C., Kim, K., Lerou, P., Daley, G.Q., 2007. Differentiation potential of histocompatible parthenogenetic embryonic stem cells. Ann. N.Y. Acad. Sci. 1106, 209–218. Li, X.J., Du, Z.W., Zarnowska, E.D., Pankratz, M., Hansen, L.O., Pearce, R.A., Zhang, S.C., 2005. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221. Liu, S., Qu, Y., Stewart, T.J., Howard, M.J., Chakrabortty, S., Holekamp, T.F., McDonald, J.W., 2000. Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation. Proc. Natl. Acad. Sci. U. S. A. 97, 6126–6131. Liu, X.Z., Xu, X.M., Hu, R., Du, C., Zhang, S.X., McDonald, J.W., Dong, H.X., Wu, Y.J., Fan, G.S., Jacquin, M.F., Hsu, C.Y., Choi, D.W., 1997. Neuronal and glial apoptosis after traumatic spinal cord injury. J. Neurosci. 17, 5395–5406. Llado, J., Haenggeli, C., Maragakis, N.J., Snyder, E.Y., Rothstein, J.D., 2004. Neural stem cells protect against glutamate-induced excitotoxicity and promote survival of injured motor neurons through the secretion of neurotrophic factors. Mol. Cell. Neurosci. 27, 322–331. Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. U. S. A. 90, 2074–2077. Lu, P., Jones, L.L., Snyder, E.Y., Tuszynski, M.H., 2003. Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury. Exp. Neurol. 181, 115–129. Lu, P., Yang, H., Jones, L.L., Filbin, M.T., Tuszynski, M.H., 2004. Combinatorial therapy with neurotrophins and cAMP promotes axonal regeneration beyond sites of spinal cord injury. J. Neurosci. 24, 6402–6409. Lu, P., Jones, L.L., Tuszynski, M.H., 2005. BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp. Neurol. 191, 344–360. Martin, G.R., 1981. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. U. S. A. 78, 7634–7638.

376

M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377

Mason, J.L., Toews, A., Hostettler, J.D., Morell, P., Suzuki, K., Goldman, J.E., Matsushima, G.K., 2004. Oligodendrocytes and progenitors become progressively depleted within chronically demyelinated lesions. Am. J. Pathol. 164, 1673–1682. Mayer-Proschel, M., Kalyani, A.J., Mujtaba, T., Rao, M.S., 1997. Isolation of lineage-restricted neuronal precursors from multipotent neuroepithelial stem cells. Neuron 19, 773–785. McTigue, D.M., Wei, P., Stokes, B.T., 2001. Proliferation of NG2-positive cells and altered oligodendrocyte numbers in the contused rat spinal cord. J. Neurosci. 21, 3392–3400. Mezey, E., Key, S., Vogelsang, G., Szalayova, I., Lange, G.D., Crain, B., 2003. Transplanted bone marrow generates new neurons in human brains. Proc. Natl. Acad. Sci. U. S. A. 100, 1364–1369. Morshead, C.M., Craig, C.G., van der Kooy, D., 1998. In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain. Development 125, 2251–2261. Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J.C., 1993. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 90, 8424–8428. Nanji, S.A., Shapiro, A.M., 2004. Islet transplantation in patients with diabetes mellitus: choice of immunosuppression. BioDrugs 18, 315–328. Nistor, G.I., Totoiu, M.O., Haque, N., Carpenter, M.K., Keirstead, H.S., 2005. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia 49, 385–396. Noel, D., Djouad, F., Bouffi, C., Mrugala, D., Jorgensen, C., 2007. Multipotent mesenchymal stromal cells and immune tolerance. Leuk. Lymphoma 48, 1283–1289. Nunes, M.C., Roy, N.S., Keyoung, H.M., Goodman, R.R., McKhann II, G., Jiang, L., Kang, J., Nedergaard, M., Goldman, S.A., 2003. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat. Med. 9, 439–447. Ogawa, Y., Sawamoto, K., Miyata, T., Miyao, S., Watanabe, M., Nakamura, M., Bregman, B.S., Koike, M., Uchiyama, Y., Toyama, Y., Okano, H., 2002. Transplantation of in vitro-expanded fetal neural progenitor cells results in neurogenesis and functional recovery after spinal cord contusion injury in adult rats. J. Neurosci. Res. 69, 925–933. Okano, H., Ogawa, Y., Nakamura, M., Kaneko, S., Iwanami, A., Toyama, Y., 2003. Transplantation of neural stem cells into the spinal cord after injury. Semin. Cell Dev. Biol. 14, 191–198. Parr, A.M., Tator, C.H., Keating, A., 2007. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant. 40, 609–619. Pastrana, E., Moreno-Flores, M.T., Gurzov, E.N., Avila, J., Wandosell, F., DiazNido, J., 2006. Genes associated with adult axon regeneration promoted by olfactory ensheathing cells: a new role for matrix metalloproteinase 2. J. Neurosci. 26, 5347–5359. Pearse, D.D., Pereira, F.C., Marcillo, A.E., Bates, M.L., Berrocal, Y.A., Filbin, M.T., Bunge, M.B., 2004. cAMP and Schwann cells promote axonal growth and functional recovery after spinal cord injury. Nat. Med. 10, 610–616. Pluchino, S., Quattrini, A., Brambilla, E., Gritti, A., Salani, G., Dina, G., Galli, R., Del Carro, U., Amadio, S., Bergami, A., Furlan, R., Comi, G., Vescovi, A.L., Martino, G., 2003. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422, 688–694. Prineas, J.W., Barnard, R.O., Kwon, E.E., Sharer, L.R., Cho, E.S., 1993. Multiple sclerosis: remyelination of nascent lesions. Ann. Neurol. 33, 137–151. Raisman, G., Li, Y., 2007. Repair of neural pathways by olfactory ensheathing cells. Nat. Rev., Neurosci. 8, 312–319. Ramer, L.M., Ramer, M.S., Steeves, J.D., 2005. Setting the stage for functional repair of spinal cord injuries: a cast of thousands. Spinal Cord 43, 134–161. Reubinoff, B.E., Itsykson, P., Turetsky, T., Pera, M.F., Reinhartz, E., Itzik, A., Ben-Hur, T., 2001. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19, 1134–1140. Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710. Richards, M., Fong, C.Y., Chan, W.K., Wong, P.C., Bongso, A., 2002. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933–936.

Richter, M.W., Roskams, A.J., 2008. Olfactory ensheathing cell transplantation following spinal cord injury: hype or hope? Exp. Neurol. 209, 353–367. Rosengard, B.R., Turka, L.A., 2001. The tolerant recipient: looking great in someone else's genes. J. Clin. Invest. 107, 33–34. Roy, N.S., Nakano, T., Keyoung, H.M., Windrem, M., Rashbaum, W.K., Alonso, M.L., Kang, J., Peng, W., Carpenter, M.K., Lin, J., Nedergaard, M., Goldman, S.A., 2004. Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord. Nat. Biotechnol. 22, 297–305. Salama, A.D., Remuzzi, G., Harmon, W.E., Sayegh, M.H., 2001. Challenges to achieving clinical transplantation tolerance. J. Clin. Invest. 108, 943–948. Sasaki, M., Honmou, O., Akiyama, Y., Uede, T., Hashi, K., Kocsis, J.D., 2001. Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 35, 26–34. Shi, J., Marinovich, A., Barres, B.A., 1998. Purification and characterization of adult oligodendrocyte precursor cells from the rat optic nerve. J. Neurosci. 18, 4627–4636. Shields, S., Gilson, J., Blakemore, W., Franklin, R., 2000. Remyelination occurs as extensively but more slowly in old rats compared to young rats following fliotoxin-induced CNS demyelination. Glia 29, 102. Shihabuddin, L.S., Horner, P.J., Ray, J., Gage, F.H., 2000. Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J. Neurosci. 20, 8727–8735. Silver, J., Miller, J.H., 2004. Regeneration beyond the glial scar. Nat. Rev. Neurosci. 5, 146–151. Snyder, E.Y., Deitcher, D.L., Walsh, C., Arnold-Aldea, S., Hartwieg, E.A., Cepko, C.L., 1992. Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 68, 33–51. Song, H., Stevens, C.F., Gage, F.H., 2002. Astroglia induce neurogenesis from adult neural stem cells. Nature 417, 39–44. Song, S., Kamath, S., Mosquera, D., Zigova, T., Sanberg, P., Vesely, D.L., Sanchez-Ramos, J., 2004. Expression of brain natriuretic peptide by human bone marrow stromal cells. Exp. Neurol. 185, 191–197. Steidl, U., Kronenwett, R., Rohr, U.P., Fenk, R., Kliszewski, S., Maercker, C., Neubert, P., Aivado, M., Koch, J., Modlich, O., Bojar, H., Gattermann, N., Haas, R., 2002. Gene expression profiling identifies significant differences between the molecular phenotypes of bone marrow-derived and circulating human CD34+ hematopoietic stem cells. Blood 99, 2037–2044. Tang, F., Shang, K., Wang, X., Gu, J., 2002. Differentiation of embryonic stem cell to astrocytes visualized by green fluorescent protein. Cell. Mol. Neurobiol. 22, 95–101. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Totoiu, M.O., Keirstead, H.S., 2005. Spinal cord injury is accompanied by chronic progressive demyelination. J. Comp. Neurol. 486, 373–383. Uchida, N., Buck, D.W., He, D., Reitsma, M.J., Masek, M., Phan, T.V., Tsukamoto, A.S., Gage, F.H., Weissman, I.L., 2000. Direct isolation of human central nervous system stem cells. Proc. Natl. Acad. Sci. U. S. A. 97, 14720–14725. Utermohlen, O., Kronke, M., 2007. Survival of priceless cells: active and passive protection of embryonic stem cells against immune destruction. Arch. Biochem. Biophys. 462, 273–277. Vrana, K.E., Hipp, J.D., Goss, A.M., McCool, B.A., Riddle, D.R., Walker, S.J., Wettstein, P.J., Studer, L.P., Tabar, V., Cunniff, K., Chapman, K., Vilner, L., West, M.D., Grant, K.A., Cibelli, J.B., 2003. Nonhuman primate parthenogenetic stem cells. Proc. Natl. Acad. Sci. U. S. A. 100 (Suppl 1), 11911–11916. Weimann, J.M., Charlton, C.A., Brazelton, T.R., Hackman, R.C., Blau, H.M., 2003. Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains. Proc. Natl. Acad. Sci. U. S. A. 100, 2088–2093. Wolswijk, G., 2000. Oligodendrocyte survival, loss and birth in lesions of chronic-stage multiple sclerosis. Brain 123 (Pt 1), 105–115. Wright, L.S., Prowse, K.R., Wallace, K., Linskens, M.H., Svendsen, C.N., 2006. Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro. Exp. Cell Res. 312, 2107–2120. Yan, J., Xu, L., Welsh, A.M., Hatfield, G., Hazel, T., Johe, K., Koliatsos, V.E., 2007. Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord. PLoS. Med. 4, e39.

M. Coutts, H.S. Keirstead / Experimental Neurology 209 (2008) 368–377 Yoon, S.H., Shim, Y.S., Park, Y.H., Chung, J.K., Nam, J.H., Kim, M.O., Park, H.C., Park, S.R., Min, B.H., Kim, E.Y., Choi, B.H., Park, H., Ha, Y., 2007. Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: phase I/II clinical trial. Stem Cells 25, 2066–2073.

377

Zeng, X., Rao, M.S., 2007. Human embryonic stem cells: long term stability, absence of senescence and a potential cell source for neural replacement. Neuroscience 145, 1348–1358. Zhang, Y.W., Denham, J., Thies, R.S., 2006. Oligodendrocyte progenitor cells derived from human embryonic stem cells express neurotrophic factors. Stem Cells Dev. 15, 943–952.