THE ANATOMICAL RECORD 293:519–530 (2010) Stem Cells: Current Approach and Future Prospects in Spinal Cord Injury Repair NING ZHANG,1 JOHANNES WIMMER,2 SHENG-JUN QIAN,1 1 AND WEI-SHAN CHEN * 1 Department of Orthopaedics, 2nd Afﬁliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of China 2 Section for Orthopedics, Campus Luebeck, University Medical Center Schleswig-Holstein, Luebeck, Germany ABSTRACT Spinal cord injury (SCI) invariably results in the loss of neurons and axonal degeneration at the lesion site, leading to permanent paralysis and loss of sensation. There has been no successful treatment for severe spinal cord injuries to recover back to normal function yet. Studies have shown that the transplantation of stem cells may provide an effective treatment for SCI because of the self-renewing and multipotential nature of these cells. Stem cells have the capability to repair injured nervous tissue through replacement of damaged cells, neuroprotection, or the creation of an environment conducive to regeneration by endogenous cells. Up to today several types of stem cells have been transplanted into the injured spinal cord. However, the question of which cell type is most beneﬁcial for SCI treatment is still unresolved. There are still several limitations to the current data sets which require further investigation. Anat C 2009 Wiley-Liss, Inc. Rec, 293:519–530, 2010. V Key words: stem cells; spinal cord injury; therapy; transplantation INTRODUCTION Spinal cord injury (SCI) invariably results in the loss of neurons and axonal degeneration at the lesion site, leading to permanent paralysis and loss of sensation below the site of the injury, because damaged nerve ﬁbers are incapable to regenerate. Unfortunately, there has been no successful treatment for severe spinal cord injuries to regain satisfactory function so far (Lim and Tow, 2007). The transplantation of stem cells may provide an effective treatment for SCI because of the selfrenewing and multipotential nature of these cells (Thuret et al., 2006). Stem cells are capable of repairing injured nervous tissue by replacing damaged cells, neuroprotection or the creation of an environment conducive to regeneration by endogenous cells. In addition, transplanted cells could offer a number of possible therapeutic uses, including delivery of therapeutic factors to provide trophic support or missing gene products, mobilization of endogenous stem cells and replacement of lost or dysfunctional cells (McDonald et al., 2004). C 2009 WILEY-LISS, INC. V Scientists are trying to use stem cells from a variety of sources to help animals with spinal cord injuries regain movement. Human embryonic and adult stem cells have been coaxed into becoming types of cells that repair damaged spinal cord insulation and replace damaged spinal cord nerve cells (Kerr et al., 2003; Li et al., 2005; Deshpande et al., 2006). To date, several types of stem cells have been transplanted into the injured spinal cord, including foetal nervous tissue, embryonic stem cells (ESCs), bone marrow mesenchymal stem cells *Correspondence to: Wei-Shan Chen, Department of Orthopaedics, 2nd Afﬁliated Hospital, School of Medicine, Zhejiang University, #88 Jiefang Road, Hangzhou, People’s Republic of China 310009. Fax: þ8657187022776. E-mail: firstname.lastname@example.org Received 9 January 2009; Accepted 21 July 2009 DOI 10.1002/ar.21025 Published online 20 November 2009 in Wiley InterScience (www.interscience.wiley.com). 520 ZHANG ET AL. (MSCs), Schwann cells, peripheral nervous tissue, collagen-based matrices containing cells, and neuroactive substances. Meanwhile, the potency of these cells and the relative ease of isolating and expanding them are invaluable properties for clinical application. Some clinical trials have also been undertaken in SCI (Daley, 2004; Grunt, 2004). THE TYPE AND FUNCTION OF STEM CELL There are two main types of stem cells which are embryonic and non-embryonic stem cells (Weissman, 2000). ESCs are totipotent and can differentiate into all three embryonic germ layers. On the other hand, non-embryonic stem cells are multipotent. Their potential to differentiate into a variety of cell types is limited to the kind of tissue where they originated from (Geuna et al., 2001; Lee and Hui, 2006). Embryonic Stem Cells As early as their derivation, ESCs have attracted a great attention to clinicians (Chen et al., 2007). Derived from early embryos, these cells remain pluripotent in culture while they can be principally expanded without any limits (Gokhan and Mehler, 2001). They give rise to most progenies and differentiate to all major somatic lineages of potential use in regenerative medicine (McDonald et al., 2004). Previous studies showed that ESCs, induced to differentiate into motor neuron and oligodendrocytes in culture, can be transplanted into damaged adult animal spinal cords to remyelinate injured neurons (Liu et al., 2000; Senior, 2000; Wichterle et al., 2002; Miles et al., 2004; Song et al., 2007). It was shown that the transplant-derived cells survived, differentiated into astrocytes, oligodendrocytes, and neurons, promoting modest functional recovery for the injured animal spinal cord. Keirstead et al. found that the human ESC-derived treatment they developed was successful in restoring the insulation tissue for neurons in rats treated 7 days after the initial SCI, which led to a recovery of motor skills. The same treatment failed on rats which had been injured for 10 months. The ﬁndings pointed to the potential of using stem cell-derived therapies for treatment of spinal cord damage in humans during the early stages of the injury (Keirstead et al., 2005). In addition, Keirstead et al. showed that transplantation of oligodendrocyte progenitor cells (OPCs) derived from human ESCs into adult rat spinal cord injuries enhanced remyelination and promoted improvement of motor function (Keirstead et al., 2005). Deshpande et al. (2006) explored the potential of motor neurons derived from ESCs to functionally replace those cells destroyed in paralyzed adult rats, demonstrating the potential of restoring functional motor units by ESCs. Recently, Cui et al. used a rat sciatic nerve transection model to show that the transplanted ESCs differentiated into myelin-forming cells and offered a potential therapy for severely injured peripheral nerves. These and other studies have clearly established the potential of ESCs transplantation for nerve repair in both the central and peripheral nervous system. Furthermore, Xie et al. (2009) showed that a combination of electrospun ﬁber scaffolds and ESCs- derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair. Multiple studies have demonstrated remyelination and motor improvement after stem cell transplantation into injured spinal cords (Nishio et al., 2006; Dasari et al., 2007b; Someya et al., 2008). However, no dramatic increase in functional recovery has been reliably demonstrated. One hypothesis suggests that muted functional recovery may be a result of improper localization, differentiation, or orientation of ESCs at the site of injection. For this reason many researchers have turned to the use of tissue scaffolds as means of structuring and organizing ESCs populations in situ. To this end, studies have investigated the optimization of ﬁbrin scaffolds for differentiation of murine ESCs into neural lineage cells and the effects of soluble growth factors on ESC differentiation inside ﬁbrin scaffolds, which could provide a new platform for the treatment of SCI (Willerth et al., 2006, 2007). Also, nanotubes, nanodevices, and polymer hygrogels have been developed to promote outgrowth and guidance of neuritis (Sykova et al., 2006b; Jan and Kotov, 2007). Despite some promising results, most of the work is still in an early stage (Xie et al., 2009). Meanwhile, a possible rejection of human ESCs following transplantation into an organ is still a challenging question. Although not yet reported, it is likely that human ESCs will express their HLA upon differentiation. Indeed, human ESCs express a low level of HLA Class I and II. The low level of HLA Class I was recently attributed to a downregulation of the antigen processing machinery (Cabrera et al., 2007). Such downregulation is often associated with tumorigenesis in cancer cells through mechanisms of immune evasion. It would allow human ESCs to be tolerated upon grafting in adult incompatible organs while raising a safety issue in the case of hyperproliferation (Puceat and Ballis, 2007). In addition, studies showed that stem cells could express low levels of MHC-I antigens and demonstrated some degree of immunotolerance (Menendez et al., 2005; Drukker et al., 2006). Non-embryonic Stem Cells Mesenchymal stem cells. Adult MSCs can be isolated from bone marrow or marrow aspirates and expanded in culture while maintaining their multipotency. These cells have two important capacities (Young et al., 2004). First, MSCs can differentiate into distinctive end-stage cell types, and have been shown by independent investigators to give rise to neural-like cells (neurons and glia) both in vitro and in vivo (Song et al., 2007). Hence, these cells can be used for reforming mesenchymal tissues through the principles and practices of tissue engineering. Preclinical studies have been performed on rats with a SCI and have shown that transplanted MSCs in the injured spinal cord survive, migrate into the host tissue and lead to axonal regeneration and motor function recovery. Dasari et al. showed that expression of caspase-3 on both neurons and oligodendrocytes after SCI was signiﬁcantly downregulated by MSC. Animals treated with MSC had higher Basso, Beattie, Bresnahan (BBB) locomotor scoring and better recovery of hind limb sensitivity. It had a positive effect on behavioral outcome and histopathological assessment after SCI (Dasari et al., 2007a). Sheth et al. showed that STEM CELLS IN SPINAL CORD INJURY REPAIR grafted human derived MSCs (hMSC) could survive for 6 weeks after transplantation, although they did not differentiate into neural or glial cells. Spinal cord injured rats grafted with hMSCs had smaller contusion cavities, which did not have a signiﬁcant inﬂuence on functional recovery (Sheth et al., 2008). Injured animals grafted with MSCs had smaller lesions 35 days post-grafting and higher scores in BBB testing than did control animals and showed a faster recovery of sensitivity in their hind limbs using the plantar test (Sykova et al., 2006b). Further clinical studies proved the safety of such an approach and partial improvement of function in patients with acute injuries (Park et al., 2005). In addition, MSCs are attractive targets for ex vivo cell and gene therapy. Ronsyn et al. investigated the feasibility of a plasmid-based strategy for genetic modiﬁcation of human hMSCs with enhanced green ﬂuorescent protein and neurotrophin (NT). They demonstrated that genetically modiﬁed hMSC lines could survive in healthy rat spinal cords over at least 3 weeks by using adequate immune suppression and can serve as vehicles for transgene expression (Ronsyn et al., 2007). Some studies have tested the feasibility of novel minimally invasive methods for transplanting MSCs into a clinically relevant spinal cord contusion model. Bakshi et al. found that MSCs delivered by lumbar puncture reached the contused spinal cord tissues and exerted a signiﬁcant beneﬁcial effect by reducing cyst and injury size. Transplantation within 14 days of injury provided signiﬁcantly greater grafting efﬁciency than a delayed delivery. An increased dosage of MSCs improved cell engraftment (Bakshi et al., 2006). Unanticipated is the realization that the MSCs secrete a large spectrum of bioactive molecules. It has also been reported that MSCs stimulate glial cells to produce colony stimulating factor (CSF), interleukins, stem cell factor, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), hepatocyte growth factor, vascular endothelial cell growth factor, neurotrophic factors such as NGF, and BDNF among others (Sykova et al., 2006b). These molecules are immunosuppressive, especially for T-cells, thus allogeneic MSCs can be considered for therapeutic use. The secreted bioactive molecules provide a regenerative microenvironment for a variety of injured adult tissues limiting the area of damage and mounting a self-regulated regenerative response. This regenerative microenvironment is referred to as trophic activity and, therefore, MSCs appear to be valuable mediators for tissue repair and regeneration. The natural titers of MSCs drawn to sites of tissue injury can be augmented by allogeneic MSCs delivered via the bloodstream (Caplan, 2007). Meanwhile, MSCs can promote axonal regeneration by guiding nerve ﬁbers (Hofstetter et al., 2002). Wu et al. (2003) showed that transplanted MSCs promote compensatory mechanisms to reorganize neural networks and activate endogenous stem cells. It was also shown that MSCs improve neurologic deﬁcits by generating either neural cells or myelin-producing cells (Sasaki et al., 2001). However, understanding the actual differentiation spectrum of stromal cells requires further investigation. Clinical studies are necessary for transferring preclinical ﬁndings from animal experiments to humans. Some studies have investigated the transplantation of unmanipulated autologous bone marrow in patients with 521 transversal SCI with respect to safety, therapeutic time window, implantation strategy, method of administration and functional improvement (Park et al., 2005; Deda et al., 2009; Vaquero and Zurita, 2009). Data has been report from 20 patients with complete SCI who received stem cell transplantation (Sykova et al., 2006a). The study showed no adverse effect that might be caused by the administration of MSCs into CSF. Although the ﬁndings of MRI after the injury suggest a poor prognosis of neural functional recovery, deﬁnite improvements were shown in motor and sensory functions up to 6 months (Saito et al., 2008). Chernykh et al. showed that transplantation of MSCs into the cyst cavity and intravenously was well tolerated, did not cause allergic or inﬂammatory reactions in the early and delayed periods after surgery, and did not induce the formation of ossiﬁcation foci in the nervous tissue. Transplantation of autologous bone marrow cells has the potential to be a novel and safe strategy for the treatment of patients in the late period after spinal trauma (Chernykh et al., 2007). In addition, Park et al. (2005) showed that autologous bone marrow cell transplantation and GM-CSF administration represent a safe protocol to efﬁciently manage SCI patients, especially those with acute complete injury. A phase I/II open-label and nonrandomized study was conducted on 35 complete SCI patients. The American Spinal Injury Association Impairment Scale (AIS) grade increased in 30.4% of the acute and subacute treated patients (AIS A–B or C), whereas no signiﬁcant improvement was observed in the chronic treatment group. Increasing neuropathic pain during the treatment and tumor formation at the site of transplantation are still remaining to be investigated (Yoon et al., 2007). More clinical trials of stem cell transplantation as treatment option for SCI are under study (Pandya, 2008). Human umbilical cord blood stem cells. Human umbilical cord blood contains a mixture of different types of stem cells in large numbers not seen in any other tissue. It is believed that human umbilical cord blood stem cells (UCBSCs) present the best alternative to ESCs as these stem cells can be used to derive tissues from the mesodermal, endodermal, and ectodermal germ lineages. In addition to being readily available, UCBSCs have more mesenchymal progenitor cells per volume, a higher pluripotent capacity and are genetically more ﬂexible than bone marrow-derived MSCs (Gang et al., 2004). Meanwhile, it has been suggested that they are not as mature as other adult stem cells so that they may not elicit alloreactive responses that modulate the immune system (Di Nicola et al., 2002; Le Blanc et al., 2003; Kang et al., 2005; Watt and Contreras, 2005). Besides these properties, UCBSCs have other distinct advantages for transplantation, including greater tolerance for HLA-mismatches between donor and recipient and a decreased risk of graft-versus-host disease (Harris, 2008). As UCBSCs have the ability to differentiate into various types of nervous cells, it may be possible to extend their application to SCI. In fact, spinal cord injured rats infused with UCBSCs showed signiﬁcant improvements 5 days post-treatment compared with untreated animals. After transplantation UCBSCs were observed at the site 522 ZHANG ET AL. of injured nervous tissue but not at uninjured regions of the spinal cord (Zhao et al., 2004). Studies examined the effects of UCBSC transplantation after complete spinal cord transection in rats. Transplanted UCBSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, NT-3, basic ﬁbroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 within the host spinal cord, which may support the repair of spinal cord injuries (Yang et al., 2008). These ﬁndings were supported by studies showing that UCBSCs transplanted into spinal cord injured animals differentiated into various neural cells, thereby improving axonal regeneration and motor function (Kuh et al., 2005). Furthermore, studies showed that systemic UCBSCs infusion signiﬁcantly attenuated SCI-induced hind limb dysfunction in rats. Serum IL-10 levels were increased while TNF-a levels were decreased after UCBSCs infusion. Both VEGF and GDNF could be detected in the injured spinal cord after transplantation of UCBSCs (Chen et al., 2008). Kao et al. reported that systemic administration of 95% pure CD34þ progenitor cells derived from UCBSCs attenuates spinal cord infarction and apoptosis as well as behavioral deﬁcits in a standard rat compression SCI model. In addition, current investigation provides new evidence suggesting that CD34þ cell therapy may cause restoration of spinal cord function during SCI by stimulating both GDNF and VEGF production in injured spinal cords (Kao et al., 2008). In previous reported clinical use of UCBSC to treat a patients with a SCI (Kang et al., 2005), it was stated that transplantation of UCBSCs improved sensory perception and mobility in hip and thigh regions. Both CT and MRI studies revealed regeneration of the spinal cord at the injury site. Dasari et al. conﬁrmed that the mechanism underlying UCBSCs neuroprotection involves activation of the Akt signaling pathway. The neuroprotective potential of UCBSCs against glutamateinduced apoptosis of cultured cortical neurons was suggested (Dasari et al., 2008b). In addition, it was indicated that human umbilical cord blood-mediated downregulation of Fas receptors and caspases leads to functional recovery of hind limbs of rats after SCI (Dasari et al., 2008a). Neural stem cells. Neural stem cells (NPCs) are potential grafts for treatment of traumatic CNS injury and neurodegenerative disorders because of their potential to differentiate into neurons and glial cells (Shihabuddin et al., 2004). Following transplantation into the injured adult rat spinal cord they survive for at least 6 weeks, migrate and maintain the ability to differentiate into the three main CNS cell lineages. Studies indicated that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and radial glia (RG), which may support axonal regeneration in future trials of transplant therapy for SCI (Kulbatski et al., 2007). However, no robust recovery in behavioral function was observed (Webber et al., 2007). Studies stated that NPCs have a distinct advantage over fetal tissue because of their greater ability to circumvent the restrictions of the blood–brain barrier and integrate throughout the central nervous system. Addi- tionally, a disease of a particular organ system is probably most efﬁciently treated with stem cells from the same organ (Daley, 2004; Snyder et al., 2004). Studies have proved that NPCs were capable of repairing damaged spinal tissue and helping to restore function in rats with spinal cord injuries (McDonald et al., 1999; Ogawa et al., 2002; Cloutier et al., 2006; Kelsch et al., 2007; Yan et al., 2007). More than one-third of the transplanted cells traveled along the spinal cord, were incorporated into damaged tissue, developed into the type of cells destroyed at the injured site and produced myelin. An injured spinal cord loses its ability to regenerate myelin-forming cells, leading to paralysis. Fehlings et al. showed that where stem cells restored myelin in the injured spine, rats showed some recovery and walked with more coordination (Karimi-Abdolrezaee et al., 2006). In an animal study, Alcon et al. grafted NPCs into the lumbar spinal cord of a mouse mutant that has a speciﬁc loss of motoneurons (progressive motor neuronopathy/pmn). A small number of grafted cells (B3000) increased the life span of the mice by 56%. The improved survival was accompanied by a rescue of host motoneurons, stabilization of weight and an increase in the size of the muscle ﬁbers. The grafted NPCs were small and round and exhibited no neural markers, suggesting that they remained in an undifferentiated state (Pan et al., 2008). Magnetically labeled NPCs were suspended with activated magnetic beads and individual NPCs, were transplanted to the co-cultures. There were few toxic effects of magnetically labeled NPCs. The differentiation potential was not changed whether NPCs were localized or scattered in vitro. Corticospinal axon growth was promoted in accordance with the transplanted NPC numbers around the organotypic co-culture. Magnetically labeled NPCs, which were directed toward the preferred site using a magnet, promoted more axon growth than scattered magnetically labeled NPCs. Overall magnetically localized labeled NPCs expressed higher potential in axon growth (Hamasaki et al., 2007). Moreover, OPCs have shown to migrate into the injured site, promoted remyelination, increased production of neurotrophic factors and improved locomotor activity, and kinematic scores in spinal cord injured rats. The study demonstrated that xenografted cells from porcine neural precursor cells might be able to recreate the damaged circuitry in CNS disease. It also showed the reliable long-term survival of grafts derived from porcine expanded neural precursors in a rat model, with maturation and integration into the host brain (El-Badri et al., 2006; Harrower et al., 2006). In addition to directly replacing damaged neurons and oligodendrocytes, stem cell therapies could also support endogenous stem cells (Thuret et al., 2006). The capacity of endogenous stem cells present in the spinal cord for regeneration is poor, and it is likely that many of the same factors that prevent axonal regeneration also inhibit the function of endogenous NPCs, including the formation of the glial scar, the lack of neurotrophic factors, inhibitory sulfated proteoglycans, and inhibitory myelinassociated molecules (Divani et al., 2007; Fitch and Silver, 2008). Nevertheless, therapies using endogenous stem cells would require no exogenous stem cell sources and would therefore circumvent the obstacle of immune rejection, as well as the ethical and moral considerations associated with their use (Bajada et al., 2008). STEM CELLS IN SPINAL CORD INJURY REPAIR Several mechanisms of central nervous system regeneration after transplantation of NPCs have been proposed (Pfeifer et al., 2006). However, the precise mechanism has not been clariﬁed. A number of studies have demonstrated that transplanted NPCs promote anatomical plasticity and modest behavioral recovery in contusive and surgical lesion models of SCI (McDonald et al., 1999; Ogawa et al., 2002). Transplanted cells may remyelinate denuded axons, decrease glial scar formation, prevent secondary cell loss, promote regeneration, form bridges and relays and replace neural cells (Lepore et al., 2005). Kamei et al. demonstrated that transplanted NPCs secreted humoral factors which in turn promoted corticospinal axon growth using the unique organotypic co-culture system involving brain cortex and spinal cord from neonatal rats (Kamei et al., 2007). The neurotrophic factors, BDNF, NT-3, and NGF, secreted by transplanted NPCs, were involved in the promotion of corticospinal axon growth after transplantation of NPCs. Axonal regeneration is inhibited by scar formation and growth-inhibitory factors associated with myelin and astrocytes (Schwab, 2004; Fawcett, 2006). Modulating the responsiveness to axonal growth-inhibitory factors and glial scar formation are attractive strategies to improve functional recovery after central nervous system injuries (Kwon et al., 2005; Freund et al., 2006; Kaneko et al., 2006; Okada et al., 2006). The majority of ependyma-derived cells differentiates into astrocyte-like cells after injury and is found in the core of the scar tissue. However, these cells are found in complementary nonoverlapping domains, which areas are immunoreactive to Chondroitin Sulfate Proteoglycans, the most important axonal growth inhibitor associated with glial scars (Busch and Silver, 2007). Moreover, axons in the scar tissue, most likely sprouts from severed axons growing into the scar tissue, were frequently found in direct proximity to ependyma-derived cells. This suggests that ependyma-derived cells in the scar tissue do not constitute a major impediment to axonal growth, but may even support some local sprouting (Meletis et al., 2008). Fetal neural progenitors present less risk of tumor formation than ESCs but inefﬁciently differentiate into motor neurons, in line with their low expression of motor neuron-speciﬁc transcription factors and poor response to soluble external factors. Bohl et al. suggested genetic engineering could drive the differentiation of fetal neural precursors into motor neurons that efﬁciently engraft in the spinal cord. To overcome this limitation, they genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1, and Neurogenin. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-speciﬁc differentiation markers and projected cholinergic axons into the ventral root (Bohl et al., 2008). Some ﬁndings suggest that co-grafts supporting NPCs may have beneﬁts for SCI. These results suggest that amniotic epithelial cells modiﬁed with the Basic Fibroblast Growth factor (bFGF) gene could enhance NPCs survival and neural differentiation in vivo and promote repair of the injured spinal cord (Meng et al., 2008). Thonhoff et al. suggested that a peptide hydrogel (PuraMatrixTM, 3DM, Cambridge, Massachusetts) was most 523 suitable for human NPC (hNPC) transplantation, because of the low toxicity, suitable gelling capability at a low concentration, gelling upon salt incorporation and permission of hNPC migration (Nisbet et al., 2008; Thonhoff et al., 2008). Atalay et al. (2007, 2008) showed that Nogo-A monoclonal antibodies (NEP1-40) promote functional recoveries in injured rat spinal cords. Pan et al. (2008) showed that synergy between Granulocate CSF (G-CSF) and neuronal stem cells may be because of the increased proliferation of progenitor cells in the injured area and increased expression of neuronal stem cell markers extrinsically or intrinsically in the distal end of injured cord. The study provided evidence that lithium may have therapeutic potential in cell replacement strategies for CNS injury because of its ability to promote proliferation and neuronal generation of grafted NPCs and reduce the host immune reaction (Su et al., 2007). In addition, there are some studies showing that Schwann cells within NPC grafts could contribute to remyelination. The NPCs present inherent plasticity to differentiate into oligodendrocytes or Schwann-like cells, depending on the host environment. Mothe and Tator (2008) stated that both cell types are capable of in the demyelinated and dysmyelinated adult spinal cord. However Schwann cells fail as a supporting platform to maintain NPCs within the graft and impair CNS axon regeneration, which makes them an unfavorable candidate for NPC graft support following SCI (Vroemen et al., 2007). PROBLEMS IN TRANSPLANTATION Although SCI could speciﬁcally beneﬁt from the engraftment of stem cells, there are some challenges facing the use of stem cells for clinical cell therapy (Baker, 2005). The ﬁrst is to develop cell-culture protocols that generate relatively deﬁned and large numbers of transplantable cells, and to obtain adequate cell survival and functionality of grafted cells after intracerebral transplantation. Secondly, potential adverse effects of transplanted stem cells, such as tumor formation, must be avoided. Grafted ESCs may generate teratocarcinomas (Erdo et al., 2003; Asano et al., 2006) and teratomas (Denker, 2006). Although differentiated progeny of ESCs are not tumorigenic, there is an absolute need to assess the tumorigenic potential of a pre-differentiated ESC culture in immune-compromised pre-clinical models before they can be considered as clinically safe (Chen et al., 2007). Finally, the risk for immune rejection of the grafted cells must be eliminated (Li et al., 2008a). Appropriate Processes One fundamental challenge facing all forms of stem cell-based therapy is that large quantities of a speciﬁc cell type must be generated, preferably without contamination of other cells that could be detrimental. Deﬁning conditions, under which a small proportion of stem cells differentiates into a desired cell type is relatively easy. It is more difﬁcult to devise strategies leading to a development of the majority of cells into a desired cell type. Phinney et al. ﬁrst demonstrated that MSCs injected into the central nervous systems of newborn mice migrate throughout the brain and adopt morphological and phenotypic characteristics of astrocytes and 524 ZHANG ET AL. neurons. Studies indicated that the methods used to promote neural cell differentiation and assess the biology of the differentiated cells are fragmented and inconsistent. Furthermore, they showed that ascribing a neural fate to MSCs is further confounded by the lack of speciﬁcity of neural markers employed, the heterogeneous nature of the MSC populations under examination, and artifacts associated with methods used to culture-expand cells in vitro (Phinney, 2007; Phinney and Prockop, 2007). The most preferable differentiation protocol for stem cells to date is the combination of the three methods: feeder cells, growth factors, and genetic engineering (Kim et al., 2006). Culture protocols based on growing stem cells together with speciﬁc feeder cells have been widely used and similar proportions of tyrosine hydroxylase (TH)-positive neurons have been obtained from mouse and human ESCs (Kawasaki et al., 2000; Barberi et al., 2003; Perrier et al., 2004; Takagi et al., 2005; Brederlau et al., 2006). Approximately 16% of murine ESCs (mESC) became TH positive when co-cultured with stromal cell feeder cells (Li et al., 2008a). Lowry et al. showed a method using a co-culture system with endothelial cells, which could improve NPC survival and preserves their multipotency, including their ability to make motor neurons. Transplantation of endothelialexpanded NPCs that were treated with sonic hedgehog (SHH) and retinoic acid (RA) during the expansion phase, into an adult mouse SCI model could result in signiﬁcant recovery of sensory and motor function (Lowry et al., 2008). Genetic manipulation is an additional strategy to improve the rate of differentiation of dopaminergic neurons from ESCs that has been tested extensively (Li et al., 2008a). For human studies, it is also important to document the karyotype of the cultured cells, although to date, cytogenetic abnormalities among passaged human MSCs are admittedly rare. Another issue that remains to be addressed is the importance of cell dosage in the transplanted inoculum and if there is a safe upper limit. Some data suggest that better functional outcomes are dose dependent. One study showed the beneﬁt of multiple injections. This may help to explain some of the varied results of different studies, but needs to be explored more extensively (Parr et al., 2007). Moreover, some studies showed that stem cells injected either intrathecally or intravenously reach the injured site at smaller concentrations and have a lower beneﬁcial effect compared with direct intraparenchymal injection (Xiao et al., 2005; Habisch et al., 2007; Niranjan et al., 2007). Timing of transplantation is also a key issue, as evidence suggests that cell survival and functional outcome may be improved if cells are transplanted at least 1 week, but no more than 14 days, after injury. The extent of cellular division after transplantation is not known. Although it has generally been suggested to be very low, this ﬁnding is not supported by all studies. At present, studies showed that the intralesional administration of bone marrow stromal cells is useful in chronic phases after SCI, in situations of established paraplegia (Zurita and Vaquero, 2004, 2006). In addition, in a study which compared the effect of systemic and local administration of MSC in adult Wistar rats suffering chronic paraplegia as consequence of severe SCI, the results showed that intravenous administration of MSC achieves some degree of functional recovery when compared with con- trol groups. Nevertheless, administration of MSC into postraumatic spinal cord cavity promotes a clear and progressive functional recovery, which is signiﬁcantly superior to the recovery obtained by means of the intravenous administration (Vaquero et al., 2006). Further studies showed that signs of functional recovery were seen 4 weeks after transplantation increasing during the following weeks (Zurita et al., 2008a,b; Vaquero and Zurita, 2009). The best method of administering MSCs is being explored. Although direct administration of MSCs results in the highest number of cells in the injury site it is an invasive procedure. Thus, intra-lesional injection may result in further local damage. Of note, cells appear to have improved survival when injected next to the lesion rather than directly into the injured area (Parr et al., 2007). As research struggles forward in the absence of federal funding, the number of stem cell lines will continue to grow, creating ever more valuable tools that are out of reach for scientists (Daley, 2004). Biomedical scientists are inherently innovators, drawn to new technologies, and these missed opportunities are difﬁcult to accept. Cell Differentiation Speciﬁcation of distinct cell types from stem cells is the key to the potential application of these naive pluripotent cells in regenerative medicine. Potential use of stem cells in biotechnology and regenerative medicine depends upon the development of strategies for directed differentiation into functional cell types. With the exception of neuroepithelial cells, which can be differentiated from hESCs with more than 95% efﬁciency (Pankratz et al., 2007), most differentiation protocols yield a mixed cell population. Differentiation to more specialized subtypes of neurons, such as midbrain dopamine neurons (Perrier et al., 2004; Yan et al., 2005; Roy et al., 2006; Sonntag et al., 2007) and spinal motor neurons (Singh Roy et al., 2005; Lee et al., 2007) becomes less efﬁcient. Consequently, it is not known what the non-target cells in the mixture are. These non-target cells are often the source of aberrant tissue formation in transplants (Brederlau et al., 2006). There is therefore a critical need to develop strategies for directed differentiation of stem cells into specialized functional cell types, such as subtypes of neural progenitors and functional motor neurons. Stem cell differentiation is amenable to manipulation by exogenous factors such as growth factors and by changes of in vitro environmental conditions (Kulbatski and Tator, 2009). In the ventral neural tube, there are ﬁve different progenitor domains (p0, p1, p2, p3, and pMN), which give rise to motoneurons and inter-neuron subtypes of the ventral spinal cord (Watt and Contreras, 2005). These progenitor domains are established mainly by interaction of Class I and Class II homeodomain proteins, which are inhibited or induced by the graded secreted inductive factors, such as SHH (Kuh et al., 2005). Previous studies showed that human stem cells can differentiate to spinal motor neurons in an adherent culture by applying retinoid acid (RA) and SHH with 20% efﬁciency (Shin et al., 2007; Soundararajan et al., 2007), similar to that from mouse ESCs. However, this efﬁciency is not ideal for a variety of analyses, and the identity of nearly 80% of the differentiated cells in the STEM CELLS IN SPINAL CORD INJURY REPAIR culture remains unknown. Present studies have developed a simple chemically deﬁned suspension culture for a near-complete restriction of hESCs to a ventral spinal progenitor fate, with highly efﬁcient generation of motor neurons. This process can also be achieved by using the small molecule purmorphamine, instead of SHH (Li et al., 2008b). To avoid debates over the point of time when a fertilized egg becomes a person, the ﬁeld has aggressively explored ethically neutral alternative sources including multipotent adult stem cells. After birth sources like the umbilical cord, placenta or amniotic ﬂuid (De Coppi et al., 2007) may provide patient-speciﬁc stem cells which could be banked at birth for autologous grafting. A promising alternative to trans-differentiation is the reprogramming of somatic cells by nuclear transfer (Hochedlinger and Jaenisch, 2002) or gene conversion (Takahashi and Yamanaka, 2006) to generate induced stem cells with properties similar to pluripotential ESCs (Okita et al., 2007; Wernig et al., 2007). Thereafter the challenge to program the differentiation of stem cells to efﬁciently generate tissue-speciﬁc cells for repair will remain (Chen et al., 2007). Though promising, such speculative cellular-engineering research does not obviate the need for expanded access to new human ESCs. Adult stem cells are not equivalent to ESCs and cannot satisfy all scientiﬁc and medical needs (Grunt, 2004). Safety The safety of stem cell derived therapy is a major issue. If, the logic goes, stem cells can regenerate the hematopoietic system of a cancer patient, then stem cells hold the promise and potential to regenerate any organ system curing various kinds of disease (Chen et al., 2007). Undifferentiated and injected in immune depressed mice, ESCs generate teratomas, a tumor made up of elements of different types of tissue. It has thus been predicted that any non-differentiated stem cell that could have escaped from a commitment or differentiation protocol may trigger a teratoma. Focused on teratomas derived from pluripotent cells, one had forgotten the possibility of tumors deriving from a progenitor cell, which could stop its differentiation and proliferate in vivo. Studies showed differences between using ESCs and adult stem cells regarding the risk of originating tumors. The clinical experience with cell therapy using adult MSCs allows clearly excluding this risk, which, however, is present when ESCs are used. Furthermore, human UCBSCs have been shown to have lower carcinogenic potential than ESCs (Kuh et al., 2005; Lim et al., 2007). Such a scenario was recently reported from neuronal progenitors grafted in rat brain (Roy et al., 2006). Of note, Puce et al. showed their experience with mice and hESCs. Committed to the cardiac lineage and engrafted in mice, sheep, and rats hESC transplantation did not reveal any kind of tumors several months after cell transplantation in infarcted myocardium (Tomescot et al., 2007). The environment that releases various soluble factors might be of importance to promote or to prevent tumor formation from hESCs. Among factors that strengthen the tumorigenic property of hESCs, several epigenetic and genetic issues must be considered (Puceat and Ballis, 2007). Chernykh et al. found that 525 transplantation of MSCs into cyst cavities and intravenously was well tolerated, did not cause any allergic or inﬂammatory reactions, and did not induce the formation of ossiﬁcation foci in the nervous tissue. Analysis of the neurological status by ASIA, Bartel, and Ashworth scales showed that in the main group the positive clinical dynamics was more often observed than in the control. The decrease in neurological deﬁcit included improvement of sensory and motor activity and conducting sensory function (Chernykh et al., 2007). Several adverse events might occur when transplanting stem cells into the spinal cord. For example, residual undifferentiated hESCs or dividing precursors might continue to proliferate in vivo and generate tumors (Draper et al., 2004). Chromosomal instability might contribute to chromosomal aberrations during long-term culturing of hESCs. This has unpredictable repercussions that obviously depend on the speciﬁc chromosomal changes. Once the cells are grafted, rapidly dividing hESCs with chromosomal changes might outgrow other cells in the transplant and promote tumor formation. Finally, hESC-derived grafts could stimulate an immune reaction in the CNS, and this might adversely affect surrounding circuitry. Vroemen et al. (2005) showed that in vivo gene expression in genetically engineered neural progenitor cells is temporally limited and mostly restricted to undifferentiated NPCs using the viral vectors tested. Removal of undifferentiated stem cells and proliferating cells before grafting is a potentially powerful approach to reduce the risk of tumor growth. Undifferentiated stem cells express unique cell-surface molecules. Upon differentiation, expression of these molecules is downregulated. Therefore, these undesired cells can be depleted by using antibodies against speciﬁc cell-surface molecules conjugated with either a ﬁuorophore or a magnetic bead in combination with ﬁuorescent activated cell sorting or magnetic activated cell sorting, respectively (Chung et al., 2006; Pruszak et al., 2007). Immunologic Barrier Li et al. (2008a) reported that cells differentiated from hESCs feature an immune privilege. In vitro immunological studies suggest that hESC-derived OPCs are poor targets for both the innate and the adaptive human immune effector cells as well as resistant to lysis by anti-Neu5Gc antibodies. These results indicate that hESC-derived OPCs retain some of the unique immunological properties of the parental cell line from which they were differentiated. However, other authors showed that OPCs derived from hESCs showed no immunoreactivity (Okamura et al., 2007). With regard to the yet controversial immunological status of stem cells, it is important to predict strategies to overcome the potential immunoincompatibility. To reach this aim, two main possibilities can be foreseen. Banking of hESCs including 150 donors with unique blood groups could provide a beneﬁcial HLA matching for most potential patients. If conﬁrmed, such a bank could be generated under Good manufacturing practice (GMP) conditions and would avoid the need of somatic cell nuclear transfer to customize hESCs, a yet not successful approach in human beings. However, 150 cell 526 ZHANG ET AL. lines may be too few to match a multiracial population of patients. The approach suggests that the differentiation potential of each cell line is the same, a concept far from being reality. Although chimerism between ESCs and recipients has been reported, another strategy to confer some immune tolerance to hESCs would be to generate tolerogenic hematopoietic cells derived from them. Together, these strategies demonstrate the possibilities to overcome the immunologic barrier (Li et al., 2008a). In the developing nervous system, netrin-1 acts as a repulsive or attractive signal, guiding nerve cells to their proper targets. In the adult spinal cord, researchers found that netrin-1 speciﬁcally repels stem cells away from the injury site, thereby preventing stem cells from replenishing nerve cells (Petit et al., 2007). In studies, HUCB cells can survive long-term in vitro and undergo neuron-like differentiation. In mice, these cells could survive no more than a month. This may relate to the differentiated state of the cells transplanted into the unlesioned striatum, rather than T cell-mediated immunological rejection (Walczak et al., 2007). Currently, immunosuppressive treatments have been widely used to inhibit immune rejection resulting from the histoincompatibility of transplanted cells. Unfortunately, they do not fully prevent chronic rejection and increase the risk of opportunistic infections (Li et al., 2008a). CONCLUSION This review has discussed the major issues associated with stem cell therapy for SCI by transplantation. Stem cells from a variety of sources have shown effectiveness in improving motor function after SCI in animal experiments and clinical trails. Differentiation and processes protocols are improving, yielding cells of higher purity and better function. Owing to their trophic and immunomodulatory properties, combining stem cell transplantation with therapies that block the activity of growth inhibitory molecules, or other conventional approaches, have the potential to be highly efﬁcacious in the treatment. Nevertheless, work remains to be done to ascertain whether these therapies can safely improve outcome after human SCI. Signiﬁcant research is still required to answer the multitude of questions that remain before these cells can proceed to further clinical trials, including the most appropriate and efﬁcacious sources of these cells, the optimal strategy and time for transplantation, and the strategy for promoting stem cell transdifferentiation and function after transplantation. Individual therapies are unlikely to emerge as a cure for SCI. Furthermore, we predict that tailored combinations of strategies will lead to cumulative improvements in outcome after different types of SCI. Clinicians, scientists, and regulatory agencies must also consider longer-term safety issues of stem cell therapies, including the likelihood of life-long immunosuppressive regimes. Meanwhile, long-term and largescale multicenter clinical studies are required to determine further the precise therapeutic effect of stem cell transplantation. ACKNOWLEDGMENT Thanks must be given to Sheng-Jie Xu PhD for critical review of the original manuscript. LITERATURE CITED Asano T, Sasaki K, Kitano Y, Terao K, Hanazono Y. 2006. In vivo tumor formation from primate embryonic stem cells. Methods Mol Biol 329:459–467. Atalay B, Bavbek M, Cekinmez M, Ozen O, Nacar A, Karabay G, Gulsen S. 2007. 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