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Neural stem cell transplantation in ALS: developing a cure for the incurable?

Neural stem cell transplantation in ALS: developing a cure for the incurable? Abstract Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease and usually causes death through respiratory muscle failure within 2–5 years of symptom onset, following progressive loss of function and considerable disability. Riluzole is currently the only disease-modifying therapeutic agent, and following recent advances in stem cell technology, the feasibility of introducing cells into the central nervous system (CNS) is being investigated. Neural stem cells (NSCs) and their derivatives have the potential to replace lost MNs and, via neurotrophic factor secretion, slow progression and stimulate regeneration through endogenous repair mechanisms. Prolonged survival and functional improvement have been demonstrated in ALS mouse models transplanted with NSCs and early clinical trials have shown NSC intraspinal injections to be safe in patients. However, issues of ethics, immunosuppression, tumour risk and technique standardization need to be addressed before NSC transplantation can become part of clinical practice. It is realistic that NSCs could be used to slow disease progression, prolong survival and even effect functional improvement in ALS, but the feasibility of stimulating CNS regeneration is questionable. This review aims to address key developments in the use of NSCs in ALS and evaluate the feasibility of their routine clinical application, with particular focus on stimulating neuronal regeneration. neural stem cells, amyotrophic lateral sclerosis, motor neuron disease, neurodegeneration, cell therapy, stem cells Introduction First described by Jean-Martin Charcot in 1869, amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder affecting an estimated 1–2 people per 100 000 worldwide (Gordon, 2013), characterized by the death of both the upper and lower motor neurons (MN) of the central nervous system (CNS). This causes spasticity, muscle weakness and atrophy, before paralysis and ultimately death as a result of respiratory muscle failure, usually within 2–5 years of symptom onset. Diagnosis is clinical and supported by electromyographic studies. The aetiology is cryptogenic in most cases, though understood to be multi-factorial. Heritability studies suggest that 60% of ALS risk can be attributed to genetics and 40% to environmental factors (Al-Chalabi et al., 2010); age, smoking and athleticism have all been associated (Huisman et al., 2011). Of note, 5–10% of cases are familial (fALS), and associated mutations have been identified in 60% (Gordon, 2013). Genes that have been implicated include Cu2+/Zn2+ superoxide dismutase (SOD1), an ubiquitous cytosolic antioxidant enzyme, TAR DNA binding protein-43 (TDP-43), involved in multiple stages of protein synthesis, and C9orf72, the protein product of which is believed to be involved in membrane trafficking. Whilst hexanucleotide repeats in C9orf72 are the most common cause of fALS (Andersen and Al-Chalabi, 2011), there are now over 160 recognized fALS-related SOD1 mutations and interestingly, similar mutations are seen in people with sporadic disease (sALS): 5% have SOD1 and 8% have C9orf72 mutations (Mackenzie et al., 2007; Millecamps et al., 2012). The identification of causative genes in fALS is largely responsible for our understanding of disease mechanisms, and much of the in vivo work exploring ALS pathogenesis has been carried out using SOD1-mutant rodent models, close mimics of ALS pathophysiology. The pathophysiological mechanisms by which these factors cause neuronal death are numerous. They include oxidative stress and subsequent mitochondrial dysfunction, membrane trafficking abnormalities, excitotoxicity (a process by which neurons are damaged by neurotransmitter excess), disruption of the cytoskeleton and axonal transport, and the aggregation of misfolded protein, as occurs in all neurodegenerative diseases (Turner and Swash, 2015). Little is known about how these pathways unite to give rise to the ALS phenotype. ALS is both rare and heterogeneous; the number and complexity of disease mechanisms present obvious challenges for developing effective therapeutic agents. Riluzole, a drug that acts to suppress glutamatergic excitotoxicity, is the only agent to date that has had disease-modifying effect, improving survival by a few months in some patients (Bucchia et al., 2015). Since the advent of riluzole in the late 1990s, more than 30 further clinical trials have yielded negative results (Aggarwal and Cudkowicz, 2008). These trials, in addition to many cellular and molecular in vitro studies, have only targeted single disease mechanisms and thus had minimal effect on disease. It is commonly accepted that any potentially impactful therapeutic agent must address many pathophysiological processes simultaneously. No such agent has yet reached clinical practice and as such, the current management of ALS is largely restricted to supportive and, inevitably, palliative care. Cell replacement therapies represent a promising therapeutic avenue; the innate plasticity of stem cells affords them the potential to modify disease in many ways (Fig. 1). Two major origins of cells are used within the context of ALS: mesenchymal stromal cells (MSCs) and cells of neural origin, of which neural stem cells (NSCs) are one type (Lunn, Sakowski and Feldman, 2014). The relative ease of obtaining MSCs makes them a particularly appealing candidate for cell replacement therapies. However, there is a lack of convincing pre-clinical data to demonstrate their long-term safety and activity in ALS models (Mazzini et al., 2003; Giordano, Galderisi and Marino, 2007; Thomsen et al., 2014), which is not the case for stem cells of neural origin. As such, this review will focus on the use of NSCs in ALS cell replacement therapy. Figure 1. Open in new tabDownload slide A schematic representation of the ways in which stem cells provide potential for modifying disease in ALS. Ideas of disease prevention and cure are speculative. NSCs have the potential to replace, support and protect neurons NSCs, the endogenous stem cells of the CNS, are self-renewing multipotent cells, able to differentiate into neurons, astrocytes (multi-functional supportive cells) and oligodendrocytes (myelinating cells of the CNS) (Temple, 1989; Cattaneo and McKay, 1990; Reynolds, Tetzlaff and Weiss, 1992) following divisions to produce further NSCs and neural progenitor cells. They can be obtained from foetal spinal cord tissue and adult brain, and more recently spinal cord, tissue by biopsy (Gage et al., 1995; Shihabuddin et al., 1995; Mothe and Tator, 2015). In the adult brain, NSCs are resident in the supraventricular zones of the lateral ventricles as well as the dentate gyrus of the hippocampus. As the predominant feature of ALS is the loss of MNs, the original premise for cell replacement therapy was direct neuronal replacement. This was despite an understanding that graft survival would be challenging. Transplanted cells would need to extend axons over considerable distances and reach their targets accurately in order to participate in functional synapses or neuromuscular junctions, a feat normally accomplished during development in utero. Further to these challenges, the toxic environment of the diseased spinal cord (a result of many of the aforementioned disease mechanisms) was likely to prevent long-term graft survival (Lunn, Sakowski and Feldman, 2014). It has also been suggested that misfolded SOD1 and TDP-43 proteins may account for ALS disease spread via a prion-like process, propagating disease from degenerating to healthy cells (Grad et al., 2011); this posed another problem for graft survival (Gordon, 2013). Symptomatic improvement exhibited following NSC transplantation in animal models of various neurodegenerative diseases has generally occurred too rapidly to be attributable to differentiation into and functional overtake of lost cells. In fact, it has now become evident that the majority of transplanted NSCs do not differentiate at all (Shihabuddin et al., 2000; Rossi and Cattaneo, 2002; Mothe et al., 2011). Thus, in ALS cell replacement therapy, MN replacement is no longer the primary aim (Lopez-Gonzalez, Knuckles and Velasco, 2009). Attention has since shifted away from the replacement of lost cells and towards the preservation of those that remain (Haidet-Phillips and Maragakis, 2015). There is growing evidence for the importance of non-neuronal cells in ALS pathogenesis, and although MN death is responsible for the clinical loss of function in ALS, many studies have shown that non-neuronal cells also become dysfunctional and can directly cause the death of healthy MNs (Redler and Dokholyan, 2012). Neurons are not solely responsible for disease, as studies in SOD1 transgenic mice have demonstrated that typical ALS did not develop if only neurons expressed the mutation (Haidet-Phillips and Maragakis, 2015). Similarly, removing the mutant gene in glia (oligodendrocytes, astrocytes and microglia) significantly improved mouse survival in another study (Haidet-Phillips and Maragakis, 2015). NSCs can differentiate into glia as well as neurons. The secretion of neurotrophic (survival) factors by glia can support damaged and at-risk neuronal populations, as well as provide functional replacement for dysfunctional endogenous glia. Not only this, but NSCs themselves can secrete trophic and anti-inflammatory factors that can modulate the extracellular environment (Ferraiuolo, Frakes and Kaspar, 2013; Haidet-Phillips and Maragakis, 2015) and protect existing neurons. It has been speculated that endogenous CNS repair mechanisms could be promoted in the same way (Fig. 1), and thus CNS remodelling and/or regeneration could be stimulated using NSCs (Teng et al., 2012; Haidet-Phillips and Maragakis, 2015). Such mechanisms are known to act in pre- and early symptomatic stages of the disease process, although regeneration occurs at a slower rate than death and degeneration. When exploring these various possible ways in which NSCs might be used to modify disease, it is also important to consider that adopting different approaches depending on the stage or pathological mechanism(s) of disease may be pertinent. Neuroprotective strategies in which cells are engineered to deliver growth factors in order to promote MN preservation, might have more effect in particular disease states than others, for which direct cell replacement may be more successful. Exactly how cell therapies might be tailored to disease subtype remains unclear and, as it will require an intricate understanding of ALS pathogenesis, is not imminent. Human NSCs have meaningful clinical effects in SOD1 transgenic rodents Following promising results from studies involving the transplantation of murine NSCs into SOD1 transgenic mice (Teng et al., 2012), attention has been turned to the use of human NSCs (hNSCs). SOD1 transgenic rats treated with foetal cord-derived hNSCs (grafted into the ventral lumbar cord) exhibited greater survival, later onset of disease and slower progression. Most hNSCs differentiated into neurons (a local two-fold increase in MN number was recorded) and formed reciprocal connections with host MNs; however, the positive effects recorded were attributed to significantly increased levels of two survival factors, glial-derived and brain-derived neurotrophic factors. It was hypothesized that neurotrophic factor secretion could itself rely upon close synaptic connections that provide optimal factor bioavailability (Xu et al., 2006). This clearly suggests that NSCs and all of their derivatives (including neurons) are important in modifying the diseased cordal milieu via neurotrophic factor secretion. A subsequent study by the same group demonstrated that an even greater extension of survival and improvement in motor function could be achieved by transplanting hNSCs into both cervical and lumbar regions of the cord, despite greater surgical trauma (Xu et al., 2011). This proof of safety and efficacy is promising, given that multiple injections would be needed were this technique to be translated into human clinical therapy in order that cervical myotomes (controlling respiratory function) were influenced. The reality of this concept has since been substantiated in a meta-analysis of 11 standardized studies of murine and hNSC (lumbar and cervical) intraspinal injections in SOD1 transgenic mice. Studies in which hNSCs were transplanted into the cervical cord showed respiratory function preservation (and prolonged survival), attributed to the conservation of MNs and their functional connections, which was evinced by estimates of motor unit number (a motor unit comprises a single MN and the skeletal muscle fibres it innervates). These studies also demonstrated safety of the technique, as function and survival were not worse in any transplanted animal compared with controls, and neither tumour formation nor inappropriate cell proliferation were detected (Teng et al., 2012). Other groups have focussed their work on manipulating NSCs specifically to secrete growth factors in order to replace glia and support MN survival, although this work could also pave the way towards understanding endogenous repair and engineering cells to be able to stimulate it. Various studies have demonstrated the success of hNSC integration into the rodent spinal cord and subsequent functional growth factor secretion following both intraspinal (Klein et al., 2005) and intrathecal injection (Hwang et al., 2009). The latter, which demonstrated the migratory capacity of hNSCs (from the CSF space to their position of integration in cord) (Hwang et al., 2009), highlights the potential for alternative, less-invasive administration techniques, although these have not been rigorously compared. Early clinical trials demonstrate safety in injecting NSCs intraspinally Following successful pre-clinical studies of hNSC transplantation in ALS models, two in-human clinical trials are currently ongoing. The first Phase 1 trial was approved by the FDA in 2009 (National Library of Medicine at the National Institutes of Health), and aimed to assess the safety of the surgical procedure, hNSC implantation itself and the necessary immunosuppressive therapy. The trial, funded by Neuralstem, involved 15 patients and employed risk-escalation methodology: the first patients involved (who also received the fewest injections) were those with the most advanced disease, as less function stood to be lost were adverse events to occur. In the initial stages, six non-ambulatory and later six ambulatory patients received lumbar intraspinal injections of foetal cord-derived hNSCs and were followed up until 18 months. Safety was demonstrated in the first (interim) analysis, published in 2012 (Glass et al., 2012), by the absence of long-term complications resulting from either the hNSCs or the procedure itself, and the tolerance by all patients (Glass et al., 2012). It was agreed that the safety of cervical injections needed to be ascertained, as these would be imperative if the treatment were to have any impact on disease progression and lifespan. In addition, dual-targeted approaches (lumbar and cervical) offered further therapeutic potential through improving both limb motor and respiratory function. Consequently, three further patients, followed by three ambulatory patients who had received lumbar injections, received cervical intraspinal injections. Results published in 2014 reported that no patients experienced acceleration of disease progression and procedural tolerance was good (Feldman et al., 2014). Preliminary results of the second Phase I trial, conducted by the Mazzini group, further evidence the safety and tolerability of lumbar intraspinal hNSC transplantation (Mazzini et al., 2015). A Phase II trial to determine safety, feasibility, toxicity and maximum hNSC dose was started in 2013 (National Library of Medicine at the National Institutes of Health). The trials have also yielded promising results relating to clinical outcomes. In the Neuralstem trial's first stage, one patient exhibited clinical improvement, and in the second stage, over 50% patients demonstrated improvements in various clinical measures, and one showed slower than expected progression (Feldman et al., 2014). In the Mazzini trial, transient functional improvements were recorded in three patients (Mazzini et al., 2015). Whilst neither trial was sufficiently powered to determine treatment efficacy, making meaningful interpretation of these findings impossible, the promise demonstrated by both is encouraging. The rate at which in-human study is occurring is also exciting. Barriers and challenges to widespread clinical translation exist Although early clinical trials have demonstrated the safety of intraspinal hNSC injections in ALS, several issues need to be addressed before clinical translation is possible. Other factors may delay clinical application. Stem cells have long been a controversial area of research. Both clinical trials involving hNSCs obtained cells from foetal material (Feldman et al., 2014; Mazzini et al., 2015), which invites considerable ethical opposition. The Mazzini group suggested that they had overcome this issue by using brain tissue from naturally miscarried foetuses (Mazzini et al., 2015); however, this is unlikely to satisfy all groups. Complex regulatory issues accompany the ethical ones, and considerable international variability still exists regarding stem cell research regulations (Eurostemcell). As a consequence of using foetal (thus non-host) cells, graft survival relies upon indefinite immunosuppression. Tacrolimus and mycophenylate mofetil (immunosuppressive agents) were the major causes of toxicity in patients on the Neuralstem trial, and most patients required dose reduction or drug cessation as a result of significant gastrointestinal side effects; these effects are well known. Whilst no major opportunistic infections were clinically manifest, three patients developed fungal infections, and one developed a basal cell (skin) carcinoma (Glass et al., 2012). It is also worth considering the potential for immunosuppression in disease potentiation, given that inflammation contributes to ALS pathophysiology (Turner and Swash, 2015). A recent development in stem cell technology could provide a solution to the issues of ethics and immunosuppression. In 2006, a group in Kyoto developed a method of generating pluripotent stem cells from adult cells by introducing genes encoding four transcription factors (Takahashi and Yamanaka, 2006). These autologous cells, commonly derived from adult fibroblasts obtained by biopsy, can divide indefinitely and differentiate to form any cell type in the human body. As the technology is relatively new, induced pluripotent stem cells (iPSCs) have mainly been used to model disease in ALS so far (Coatti et al., 2015). However, a limited number of studies have investigated therapeutic applications. One such study, in which iPSC-derived NSCs were intrathecally and intravenously transplanted into ALS mouse models, demonstrated functional improvement and prolonged survival as a result of neurotrophic factor secretion and a reduction in microglial activity. These results provide evidence for both the efficacy and safety of iPSC-derived NSCs, as no abnormal cell proliferation and, importantly, no tumour growth were detected in transplant recipients (Nizzardo et al., 2014). Tumour formation is a risk that accompanies the use of all types of stem cell (Teng et al., 2012), and whilst they hold huge potential for future developments in cell replacement therapies, iPSCs have a greater propensity towards tumour formation than other stem cell types; the risk is significant (Lu and Zhao, 2013; Coatti et al., 2015). Whilst iPSCs could overcome ethical and immunosuppressive barriers to the clinical translation of NSC therapy, it would be counterproductive to replace one devastating disease with another. It is also worth noting that the challenge of standardizing transplant methodology may delay the therapeutic application of NSCs in ALS, as each method will require individual and comparative trialling (Lunn, Sakowski and Feldman, 2014). This applies to both cell derivation and surgical techniques, several of which have already been mentioned (Hwang et al., 2009, Nizzardo et al., 2014; Feldman et al., 2014). Conclusion The potential of NSCs to prolong survival and produce functional improvement in ALS has been widely evidenced in animal models. The results of early clinical trials are promising and demonstrate the safety of NSC transplantation in humans. Considerable progress has been made during the past decade, and so it is not unrealistic to expect NSC transplantation to reach clinical practice within the next 15 years, following successful completion of Phase III trials. Whether or not this could become routine is less certain, as many challenges still exist. Whilst NSCs can feasibly slow degeneration and provide neuroprotection, stimulating CNS regeneration is less realistic in the near future. To make this possible, further research is needed not only to understand endogenous repair mechanisms sufficiently to be able to stimulate them, but then also to enable full and safe control of regenerative processes. Unfortunately, NSCs are unlikely to constitute a cure for ALS. Author biography Amy is a medical student at King's College London in her final year of study. She recently completed an intercalated BSc in Neuroscience and Mental Health at Imperial College London with First Class Honours, and was awarded the Malcolm Morris Memorial Prize for best academic performance on the course. She has a keen interest in all areas of neuroscience and aspires to a career combining both clinical and academic neurology. Previous work has included developing a database for a regional Myasthenia Gravis service, and an audit of seizure presentations to a District General Hospital Emergency Department over a 2-year period. The latter was selected for a platform presentation at the Association of British Neurologists Annual Meeting in 2015, where Amy was awarded the Charles Symonds Prize for best audit. Amy was also the recipient of the 2015 Gowers’ Medical Student Award for an essay based upon this epilepsy audit. Outside of medicine, Amy is a member of the University of London Symphony Orchestra and holds DipABRSM with Distinction in viola performance. 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Neural stem cell transplantation in ALS: developing a cure for the incurable?

BioScience Horizons , Volume 9 – Jan 1, 2016

Neural stem cell transplantation in ALS: developing a cure for the incurable?

BioScience Horizons , Volume 9 – Jan 1, 2016

Abstract

Abstract Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease and usually causes death through respiratory muscle failure within 2–5 years of symptom onset, following progressive loss of function and considerable disability. Riluzole is currently the only disease-modifying therapeutic agent, and following recent advances in stem cell technology, the feasibility of introducing cells into the central nervous system (CNS) is being investigated. Neural stem cells (NSCs) and their derivatives have the potential to replace lost MNs and, via neurotrophic factor secretion, slow progression and stimulate regeneration through endogenous repair mechanisms. Prolonged survival and functional improvement have been demonstrated in ALS mouse models transplanted with NSCs and early clinical trials have shown NSC intraspinal injections to be safe in patients. However, issues of ethics, immunosuppression, tumour risk and technique standardization need to be addressed before NSC transplantation can become part of clinical practice. It is realistic that NSCs could be used to slow disease progression, prolong survival and even effect functional improvement in ALS, but the feasibility of stimulating CNS regeneration is questionable. This review aims to address key developments in the use of NSCs in ALS and evaluate the feasibility of their routine clinical application, with particular focus on stimulating neuronal regeneration. neural stem cells, amyotrophic lateral sclerosis, motor neuron disease, neurodegeneration, cell therapy, stem cells Introduction First described by Jean-Martin Charcot in 1869, amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder affecting an estimated 1–2 people per 100 000 worldwide (Gordon, 2013), characterized by the death of both the upper and lower motor neurons (MN) of the central nervous system (CNS). This causes spasticity, muscle weakness and atrophy, before paralysis and ultimately death as a result of respiratory muscle failure, usually within 2–5 years of symptom onset. Diagnosis is clinical and supported by electromyographic studies. The aetiology is cryptogenic in most cases, though understood to be multi-factorial. Heritability studies suggest that 60% of ALS risk can be attributed to genetics and 40% to environmental factors (Al-Chalabi et al., 2010); age, smoking and athleticism have all been associated (Huisman et al., 2011). Of note, 5–10% of cases are familial (fALS), and associated mutations have been identified in 60% (Gordon, 2013). Genes that have been implicated include Cu2+/Zn2+ superoxide dismutase (SOD1), an ubiquitous cytosolic antioxidant enzyme, TAR DNA binding protein-43 (TDP-43), involved in multiple stages of protein synthesis, and C9orf72, the protein product of which is believed to be involved in membrane trafficking. Whilst hexanucleotide repeats in C9orf72 are the most common cause of fALS (Andersen and Al-Chalabi, 2011), there are now over 160 recognized fALS-related SOD1 mutations and interestingly, similar mutations are seen in people with sporadic disease (sALS): 5% have SOD1 and 8% have C9orf72 mutations (Mackenzie et al., 2007; Millecamps et al., 2012). The identification of causative genes in fALS is largely responsible for our understanding of disease mechanisms, and much of the in vivo work exploring ALS pathogenesis has been carried out using SOD1-mutant rodent models, close mimics of ALS pathophysiology. The pathophysiological mechanisms by which these factors cause neuronal death are numerous. They include oxidative stress and subsequent mitochondrial dysfunction, membrane trafficking abnormalities, excitotoxicity (a process by which neurons are damaged by neurotransmitter excess), disruption of the cytoskeleton and axonal transport, and the aggregation of misfolded protein, as occurs in all neurodegenerative diseases (Turner and Swash, 2015). Little is known about how these pathways unite to give rise to the ALS phenotype. ALS is both rare and heterogeneous; the number and complexity of disease mechanisms present obvious challenges for developing effective therapeutic agents. Riluzole, a drug that acts to suppress glutamatergic excitotoxicity, is the only agent to date that has had disease-modifying effect, improving survival by a few months in some patients (Bucchia et al., 2015). Since the advent of riluzole in the late 1990s, more than 30 further clinical trials have yielded negative results (Aggarwal and Cudkowicz, 2008). These trials, in addition to many cellular and molecular in vitro studies, have only targeted single disease mechanisms and thus had minimal effect on disease. It is commonly accepted that any potentially impactful therapeutic agent must address many pathophysiological processes simultaneously. No such agent has yet reached clinical practice and as such, the current management of ALS is largely restricted to supportive and, inevitably, palliative care. Cell replacement therapies represent a promising therapeutic avenue; the innate plasticity of stem cells affords them the potential to modify disease in many ways (Fig. 1). Two major origins of cells are used within the context of ALS: mesenchymal stromal cells (MSCs) and cells of neural origin, of which neural stem cells (NSCs) are one type (Lunn, Sakowski and Feldman, 2014). The relative ease of obtaining MSCs makes them a particularly appealing candidate for cell replacement therapies. However, there is a lack of convincing pre-clinical data to demonstrate their long-term safety and activity in ALS models (Mazzini et al., 2003; Giordano, Galderisi and Marino, 2007; Thomsen et al., 2014), which is not the case for stem cells of neural origin. As such, this review will focus on the use of NSCs in ALS cell replacement therapy. Figure 1. Open in new tabDownload slide A schematic representation of the ways in which stem cells provide potential for modifying disease in ALS. Ideas of disease prevention and cure are speculative. NSCs have the potential to replace, support and protect neurons NSCs, the endogenous stem cells of the CNS, are self-renewing multipotent cells, able to differentiate into neurons, astrocytes (multi-functional supportive cells) and oligodendrocytes (myelinating cells of the CNS) (Temple, 1989; Cattaneo and McKay, 1990; Reynolds, Tetzlaff and Weiss, 1992) following divisions to produce further NSCs and neural progenitor cells. They can be obtained from foetal spinal cord tissue and adult brain, and more recently spinal cord, tissue by biopsy (Gage et al., 1995; Shihabuddin et al., 1995; Mothe and Tator, 2015). In the adult brain, NSCs are resident in the supraventricular zones of the lateral ventricles as well as the dentate gyrus of the hippocampus. As the predominant feature of ALS is the loss of MNs, the original premise for cell replacement therapy was direct neuronal replacement. This was despite an understanding that graft survival would be challenging. Transplanted cells would need to extend axons over considerable distances and reach their targets accurately in order to participate in functional synapses or neuromuscular junctions, a feat normally accomplished during development in utero. Further to these challenges, the toxic environment of the diseased spinal cord (a result of many of the aforementioned disease mechanisms) was likely to prevent long-term graft survival (Lunn, Sakowski and Feldman, 2014). It has also been suggested that misfolded SOD1 and TDP-43 proteins may account for ALS disease spread via a prion-like process, propagating disease from degenerating to healthy cells (Grad et al., 2011); this posed another problem for graft survival (Gordon, 2013). Symptomatic improvement exhibited following NSC transplantation in animal models of various neurodegenerative diseases has generally occurred too rapidly to be attributable to differentiation into and functional overtake of lost cells. In fact, it has now become evident that the majority of transplanted NSCs do not differentiate at all (Shihabuddin et al., 2000; Rossi and Cattaneo, 2002; Mothe et al., 2011). Thus, in ALS cell replacement therapy, MN replacement is no longer the primary aim (Lopez-Gonzalez, Knuckles and Velasco, 2009). Attention has since shifted away from the replacement of lost cells and towards the preservation of those that remain (Haidet-Phillips and Maragakis, 2015). There is growing evidence for the importance of non-neuronal cells in ALS pathogenesis, and although MN death is responsible for the clinical loss of function in ALS, many studies have shown that non-neuronal cells also become dysfunctional and can directly cause the death of healthy MNs (Redler and Dokholyan, 2012). Neurons are not solely responsible for disease, as studies in SOD1 transgenic mice have demonstrated that typical ALS did not develop if only neurons expressed the mutation (Haidet-Phillips and Maragakis, 2015). Similarly, removing the mutant gene in glia (oligodendrocytes, astrocytes and microglia) significantly improved mouse survival in another study (Haidet-Phillips and Maragakis, 2015). NSCs can differentiate into glia as well as neurons. The secretion of neurotrophic (survival) factors by glia can support damaged and at-risk neuronal populations, as well as provide functional replacement for dysfunctional endogenous glia. Not only this, but NSCs themselves can secrete trophic and anti-inflammatory factors that can modulate the extracellular environment (Ferraiuolo, Frakes and Kaspar, 2013; Haidet-Phillips and Maragakis, 2015) and protect existing neurons. It has been speculated that endogenous CNS repair mechanisms could be promoted in the same way (Fig. 1), and thus CNS remodelling and/or regeneration could be stimulated using NSCs (Teng et al., 2012; Haidet-Phillips and Maragakis, 2015). Such mechanisms are known to act in pre- and early symptomatic stages of the disease process, although regeneration occurs at a slower rate than death and degeneration. When exploring these various possible ways in which NSCs might be used to modify disease, it is also important to consider that adopting different approaches depending on the stage or pathological mechanism(s) of disease may be pertinent. Neuroprotective strategies in which cells are engineered to deliver growth factors in order to promote MN preservation, might have more effect in particular disease states than others, for which direct cell replacement may be more successful. Exactly how cell therapies might be tailored to disease subtype remains unclear and, as it will require an intricate understanding of ALS pathogenesis, is not imminent. Human NSCs have meaningful clinical effects in SOD1 transgenic rodents Following promising results from studies involving the transplantation of murine NSCs into SOD1 transgenic mice (Teng et al., 2012), attention has been turned to the use of human NSCs (hNSCs). SOD1 transgenic rats treated with foetal cord-derived hNSCs (grafted into the ventral lumbar cord) exhibited greater survival, later onset of disease and slower progression. Most hNSCs differentiated into neurons (a local two-fold increase in MN number was recorded) and formed reciprocal connections with host MNs; however, the positive effects recorded were attributed to significantly increased levels of two survival factors, glial-derived and brain-derived neurotrophic factors. It was hypothesized that neurotrophic factor secretion could itself rely upon close synaptic connections that provide optimal factor bioavailability (Xu et al., 2006). This clearly suggests that NSCs and all of their derivatives (including neurons) are important in modifying the diseased cordal milieu via neurotrophic factor secretion. A subsequent study by the same group demonstrated that an even greater extension of survival and improvement in motor function could be achieved by transplanting hNSCs into both cervical and lumbar regions of the cord, despite greater surgical trauma (Xu et al., 2011). This proof of safety and efficacy is promising, given that multiple injections would be needed were this technique to be translated into human clinical therapy in order that cervical myotomes (controlling respiratory function) were influenced. The reality of this concept has since been substantiated in a meta-analysis of 11 standardized studies of murine and hNSC (lumbar and cervical) intraspinal injections in SOD1 transgenic mice. Studies in which hNSCs were transplanted into the cervical cord showed respiratory function preservation (and prolonged survival), attributed to the conservation of MNs and their functional connections, which was evinced by estimates of motor unit number (a motor unit comprises a single MN and the skeletal muscle fibres it innervates). These studies also demonstrated safety of the technique, as function and survival were not worse in any transplanted animal compared with controls, and neither tumour formation nor inappropriate cell proliferation were detected (Teng et al., 2012). Other groups have focussed their work on manipulating NSCs specifically to secrete growth factors in order to replace glia and support MN survival, although this work could also pave the way towards understanding endogenous repair and engineering cells to be able to stimulate it. Various studies have demonstrated the success of hNSC integration into the rodent spinal cord and subsequent functional growth factor secretion following both intraspinal (Klein et al., 2005) and intrathecal injection (Hwang et al., 2009). The latter, which demonstrated the migratory capacity of hNSCs (from the CSF space to their position of integration in cord) (Hwang et al., 2009), highlights the potential for alternative, less-invasive administration techniques, although these have not been rigorously compared. Early clinical trials demonstrate safety in injecting NSCs intraspinally Following successful pre-clinical studies of hNSC transplantation in ALS models, two in-human clinical trials are currently ongoing. The first Phase 1 trial was approved by the FDA in 2009 (National Library of Medicine at the National Institutes of Health), and aimed to assess the safety of the surgical procedure, hNSC implantation itself and the necessary immunosuppressive therapy. The trial, funded by Neuralstem, involved 15 patients and employed risk-escalation methodology: the first patients involved (who also received the fewest injections) were those with the most advanced disease, as less function stood to be lost were adverse events to occur. In the initial stages, six non-ambulatory and later six ambulatory patients received lumbar intraspinal injections of foetal cord-derived hNSCs and were followed up until 18 months. Safety was demonstrated in the first (interim) analysis, published in 2012 (Glass et al., 2012), by the absence of long-term complications resulting from either the hNSCs or the procedure itself, and the tolerance by all patients (Glass et al., 2012). It was agreed that the safety of cervical injections needed to be ascertained, as these would be imperative if the treatment were to have any impact on disease progression and lifespan. In addition, dual-targeted approaches (lumbar and cervical) offered further therapeutic potential through improving both limb motor and respiratory function. Consequently, three further patients, followed by three ambulatory patients who had received lumbar injections, received cervical intraspinal injections. Results published in 2014 reported that no patients experienced acceleration of disease progression and procedural tolerance was good (Feldman et al., 2014). Preliminary results of the second Phase I trial, conducted by the Mazzini group, further evidence the safety and tolerability of lumbar intraspinal hNSC transplantation (Mazzini et al., 2015). A Phase II trial to determine safety, feasibility, toxicity and maximum hNSC dose was started in 2013 (National Library of Medicine at the National Institutes of Health). The trials have also yielded promising results relating to clinical outcomes. In the Neuralstem trial's first stage, one patient exhibited clinical improvement, and in the second stage, over 50% patients demonstrated improvements in various clinical measures, and one showed slower than expected progression (Feldman et al., 2014). In the Mazzini trial, transient functional improvements were recorded in three patients (Mazzini et al., 2015). Whilst neither trial was sufficiently powered to determine treatment efficacy, making meaningful interpretation of these findings impossible, the promise demonstrated by both is encouraging. The rate at which in-human study is occurring is also exciting. Barriers and challenges to widespread clinical translation exist Although early clinical trials have demonstrated the safety of intraspinal hNSC injections in ALS, several issues need to be addressed before clinical translation is possible. Other factors may delay clinical application. Stem cells have long been a controversial area of research. Both clinical trials involving hNSCs obtained cells from foetal material (Feldman et al., 2014; Mazzini et al., 2015), which invites considerable ethical opposition. The Mazzini group suggested that they had overcome this issue by using brain tissue from naturally miscarried foetuses (Mazzini et al., 2015); however, this is unlikely to satisfy all groups. Complex regulatory issues accompany the ethical ones, and considerable international variability still exists regarding stem cell research regulations (Eurostemcell). As a consequence of using foetal (thus non-host) cells, graft survival relies upon indefinite immunosuppression. Tacrolimus and mycophenylate mofetil (immunosuppressive agents) were the major causes of toxicity in patients on the Neuralstem trial, and most patients required dose reduction or drug cessation as a result of significant gastrointestinal side effects; these effects are well known. Whilst no major opportunistic infections were clinically manifest, three patients developed fungal infections, and one developed a basal cell (skin) carcinoma (Glass et al., 2012). It is also worth considering the potential for immunosuppression in disease potentiation, given that inflammation contributes to ALS pathophysiology (Turner and Swash, 2015). A recent development in stem cell technology could provide a solution to the issues of ethics and immunosuppression. In 2006, a group in Kyoto developed a method of generating pluripotent stem cells from adult cells by introducing genes encoding four transcription factors (Takahashi and Yamanaka, 2006). These autologous cells, commonly derived from adult fibroblasts obtained by biopsy, can divide indefinitely and differentiate to form any cell type in the human body. As the technology is relatively new, induced pluripotent stem cells (iPSCs) have mainly been used to model disease in ALS so far (Coatti et al., 2015). However, a limited number of studies have investigated therapeutic applications. One such study, in which iPSC-derived NSCs were intrathecally and intravenously transplanted into ALS mouse models, demonstrated functional improvement and prolonged survival as a result of neurotrophic factor secretion and a reduction in microglial activity. These results provide evidence for both the efficacy and safety of iPSC-derived NSCs, as no abnormal cell proliferation and, importantly, no tumour growth were detected in transplant recipients (Nizzardo et al., 2014). Tumour formation is a risk that accompanies the use of all types of stem cell (Teng et al., 2012), and whilst they hold huge potential for future developments in cell replacement therapies, iPSCs have a greater propensity towards tumour formation than other stem cell types; the risk is significant (Lu and Zhao, 2013; Coatti et al., 2015). Whilst iPSCs could overcome ethical and immunosuppressive barriers to the clinical translation of NSC therapy, it would be counterproductive to replace one devastating disease with another. It is also worth noting that the challenge of standardizing transplant methodology may delay the therapeutic application of NSCs in ALS, as each method will require individual and comparative trialling (Lunn, Sakowski and Feldman, 2014). This applies to both cell derivation and surgical techniques, several of which have already been mentioned (Hwang et al., 2009, Nizzardo et al., 2014; Feldman et al., 2014). Conclusion The potential of NSCs to prolong survival and produce functional improvement in ALS has been widely evidenced in animal models. The results of early clinical trials are promising and demonstrate the safety of NSC transplantation in humans. Considerable progress has been made during the past decade, and so it is not unrealistic to expect NSC transplantation to reach clinical practice within the next 15 years, following successful completion of Phase III trials. Whether or not this could become routine is less certain, as many challenges still exist. Whilst NSCs can feasibly slow degeneration and provide neuroprotection, stimulating CNS regeneration is less realistic in the near future. To make this possible, further research is needed not only to understand endogenous repair mechanisms sufficiently to be able to stimulate them, but then also to enable full and safe control of regenerative processes. Unfortunately, NSCs are unlikely to constitute a cure for ALS. Author biography Amy is a medical student at King's College London in her final year of study. She recently completed an intercalated BSc in Neuroscience and Mental Health at Imperial College London with First Class Honours, and was awarded the Malcolm Morris Memorial Prize for best academic performance on the course. She has a keen interest in all areas of neuroscience and aspires to a career combining both clinical and academic neurology. Previous work has included developing a database for a regional Myasthenia Gravis service, and an audit of seizure presentations to a District General Hospital Emergency Department over a 2-year period. The latter was selected for a platform presentation at the Association of British Neurologists Annual Meeting in 2015, where Amy was awarded the Charles Symonds Prize for best audit. Amy was also the recipient of the 2015 Gowers’ Medical Student Award for an essay based upon this epilepsy audit. Outside of medicine, Amy is a member of the University of London Symphony Orchestra and holds DipABRSM with Distinction in viola performance. 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( 2006 ) Human neural stem cell grafts ameliorate motor neuron disease in SOD-1 transgenic rats , Transplantation , 82 ( 7 ), 865 – 875 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Supervisor: Dr Jane Saffell, Institute of Medical & Biomedical Education, St. George's University of London, Cranmer Terrace, London, SW17 0RE, UK. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author 2016. Published by Oxford University Press.

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Abstract

Abstract Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease and usually causes death through respiratory muscle failure within 2–5 years of symptom onset, following progressive loss of function and considerable disability. Riluzole is currently the only disease-modifying therapeutic agent, and following recent advances in stem cell technology, the feasibility of introducing cells into the central nervous system (CNS) is being investigated. Neural stem cells (NSCs) and their derivatives have the potential to replace lost MNs and, via neurotrophic factor secretion, slow progression and stimulate regeneration through endogenous repair mechanisms. Prolonged survival and functional improvement have been demonstrated in ALS mouse models transplanted with NSCs and early clinical trials have shown NSC intraspinal injections to be safe in patients. However, issues of ethics, immunosuppression, tumour risk and technique standardization need to be addressed before NSC transplantation can become part of clinical practice. It is realistic that NSCs could be used to slow disease progression, prolong survival and even effect functional improvement in ALS, but the feasibility of stimulating CNS regeneration is questionable. This review aims to address key developments in the use of NSCs in ALS and evaluate the feasibility of their routine clinical application, with particular focus on stimulating neuronal regeneration. neural stem cells, amyotrophic lateral sclerosis, motor neuron disease, neurodegeneration, cell therapy, stem cells Introduction First described by Jean-Martin Charcot in 1869, amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder affecting an estimated 1–2 people per 100 000 worldwide (Gordon, 2013), characterized by the death of both the upper and lower motor neurons (MN) of the central nervous system (CNS). This causes spasticity, muscle weakness and atrophy, before paralysis and ultimately death as a result of respiratory muscle failure, usually within 2–5 years of symptom onset. Diagnosis is clinical and supported by electromyographic studies. The aetiology is cryptogenic in most cases, though understood to be multi-factorial. Heritability studies suggest that 60% of ALS risk can be attributed to genetics and 40% to environmental factors (Al-Chalabi et al., 2010); age, smoking and athleticism have all been associated (Huisman et al., 2011). Of note, 5–10% of cases are familial (fALS), and associated mutations have been identified in 60% (Gordon, 2013). Genes that have been implicated include Cu2+/Zn2+ superoxide dismutase (SOD1), an ubiquitous cytosolic antioxidant enzyme, TAR DNA binding protein-43 (TDP-43), involved in multiple stages of protein synthesis, and C9orf72, the protein product of which is believed to be involved in membrane trafficking. Whilst hexanucleotide repeats in C9orf72 are the most common cause of fALS (Andersen and Al-Chalabi, 2011), there are now over 160 recognized fALS-related SOD1 mutations and interestingly, similar mutations are seen in people with sporadic disease (sALS): 5% have SOD1 and 8% have C9orf72 mutations (Mackenzie et al., 2007; Millecamps et al., 2012). The identification of causative genes in fALS is largely responsible for our understanding of disease mechanisms, and much of the in vivo work exploring ALS pathogenesis has been carried out using SOD1-mutant rodent models, close mimics of ALS pathophysiology. The pathophysiological mechanisms by which these factors cause neuronal death are numerous. They include oxidative stress and subsequent mitochondrial dysfunction, membrane trafficking abnormalities, excitotoxicity (a process by which neurons are damaged by neurotransmitter excess), disruption of the cytoskeleton and axonal transport, and the aggregation of misfolded protein, as occurs in all neurodegenerative diseases (Turner and Swash, 2015). Little is known about how these pathways unite to give rise to the ALS phenotype. ALS is both rare and heterogeneous; the number and complexity of disease mechanisms present obvious challenges for developing effective therapeutic agents. Riluzole, a drug that acts to suppress glutamatergic excitotoxicity, is the only agent to date that has had disease-modifying effect, improving survival by a few months in some patients (Bucchia et al., 2015). Since the advent of riluzole in the late 1990s, more than 30 further clinical trials have yielded negative results (Aggarwal and Cudkowicz, 2008). These trials, in addition to many cellular and molecular in vitro studies, have only targeted single disease mechanisms and thus had minimal effect on disease. It is commonly accepted that any potentially impactful therapeutic agent must address many pathophysiological processes simultaneously. No such agent has yet reached clinical practice and as such, the current management of ALS is largely restricted to supportive and, inevitably, palliative care. Cell replacement therapies represent a promising therapeutic avenue; the innate plasticity of stem cells affords them the potential to modify disease in many ways (Fig. 1). Two major origins of cells are used within the context of ALS: mesenchymal stromal cells (MSCs) and cells of neural origin, of which neural stem cells (NSCs) are one type (Lunn, Sakowski and Feldman, 2014). The relative ease of obtaining MSCs makes them a particularly appealing candidate for cell replacement therapies. However, there is a lack of convincing pre-clinical data to demonstrate their long-term safety and activity in ALS models (Mazzini et al., 2003; Giordano, Galderisi and Marino, 2007; Thomsen et al., 2014), which is not the case for stem cells of neural origin. As such, this review will focus on the use of NSCs in ALS cell replacement therapy. Figure 1. Open in new tabDownload slide A schematic representation of the ways in which stem cells provide potential for modifying disease in ALS. Ideas of disease prevention and cure are speculative. NSCs have the potential to replace, support and protect neurons NSCs, the endogenous stem cells of the CNS, are self-renewing multipotent cells, able to differentiate into neurons, astrocytes (multi-functional supportive cells) and oligodendrocytes (myelinating cells of the CNS) (Temple, 1989; Cattaneo and McKay, 1990; Reynolds, Tetzlaff and Weiss, 1992) following divisions to produce further NSCs and neural progenitor cells. They can be obtained from foetal spinal cord tissue and adult brain, and more recently spinal cord, tissue by biopsy (Gage et al., 1995; Shihabuddin et al., 1995; Mothe and Tator, 2015). In the adult brain, NSCs are resident in the supraventricular zones of the lateral ventricles as well as the dentate gyrus of the hippocampus. As the predominant feature of ALS is the loss of MNs, the original premise for cell replacement therapy was direct neuronal replacement. This was despite an understanding that graft survival would be challenging. Transplanted cells would need to extend axons over considerable distances and reach their targets accurately in order to participate in functional synapses or neuromuscular junctions, a feat normally accomplished during development in utero. Further to these challenges, the toxic environment of the diseased spinal cord (a result of many of the aforementioned disease mechanisms) was likely to prevent long-term graft survival (Lunn, Sakowski and Feldman, 2014). It has also been suggested that misfolded SOD1 and TDP-43 proteins may account for ALS disease spread via a prion-like process, propagating disease from degenerating to healthy cells (Grad et al., 2011); this posed another problem for graft survival (Gordon, 2013). Symptomatic improvement exhibited following NSC transplantation in animal models of various neurodegenerative diseases has generally occurred too rapidly to be attributable to differentiation into and functional overtake of lost cells. In fact, it has now become evident that the majority of transplanted NSCs do not differentiate at all (Shihabuddin et al., 2000; Rossi and Cattaneo, 2002; Mothe et al., 2011). Thus, in ALS cell replacement therapy, MN replacement is no longer the primary aim (Lopez-Gonzalez, Knuckles and Velasco, 2009). Attention has since shifted away from the replacement of lost cells and towards the preservation of those that remain (Haidet-Phillips and Maragakis, 2015). There is growing evidence for the importance of non-neuronal cells in ALS pathogenesis, and although MN death is responsible for the clinical loss of function in ALS, many studies have shown that non-neuronal cells also become dysfunctional and can directly cause the death of healthy MNs (Redler and Dokholyan, 2012). Neurons are not solely responsible for disease, as studies in SOD1 transgenic mice have demonstrated that typical ALS did not develop if only neurons expressed the mutation (Haidet-Phillips and Maragakis, 2015). Similarly, removing the mutant gene in glia (oligodendrocytes, astrocytes and microglia) significantly improved mouse survival in another study (Haidet-Phillips and Maragakis, 2015). NSCs can differentiate into glia as well as neurons. The secretion of neurotrophic (survival) factors by glia can support damaged and at-risk neuronal populations, as well as provide functional replacement for dysfunctional endogenous glia. Not only this, but NSCs themselves can secrete trophic and anti-inflammatory factors that can modulate the extracellular environment (Ferraiuolo, Frakes and Kaspar, 2013; Haidet-Phillips and Maragakis, 2015) and protect existing neurons. It has been speculated that endogenous CNS repair mechanisms could be promoted in the same way (Fig. 1), and thus CNS remodelling and/or regeneration could be stimulated using NSCs (Teng et al., 2012; Haidet-Phillips and Maragakis, 2015). Such mechanisms are known to act in pre- and early symptomatic stages of the disease process, although regeneration occurs at a slower rate than death and degeneration. When exploring these various possible ways in which NSCs might be used to modify disease, it is also important to consider that adopting different approaches depending on the stage or pathological mechanism(s) of disease may be pertinent. Neuroprotective strategies in which cells are engineered to deliver growth factors in order to promote MN preservation, might have more effect in particular disease states than others, for which direct cell replacement may be more successful. Exactly how cell therapies might be tailored to disease subtype remains unclear and, as it will require an intricate understanding of ALS pathogenesis, is not imminent. Human NSCs have meaningful clinical effects in SOD1 transgenic rodents Following promising results from studies involving the transplantation of murine NSCs into SOD1 transgenic mice (Teng et al., 2012), attention has been turned to the use of human NSCs (hNSCs). SOD1 transgenic rats treated with foetal cord-derived hNSCs (grafted into the ventral lumbar cord) exhibited greater survival, later onset of disease and slower progression. Most hNSCs differentiated into neurons (a local two-fold increase in MN number was recorded) and formed reciprocal connections with host MNs; however, the positive effects recorded were attributed to significantly increased levels of two survival factors, glial-derived and brain-derived neurotrophic factors. It was hypothesized that neurotrophic factor secretion could itself rely upon close synaptic connections that provide optimal factor bioavailability (Xu et al., 2006). This clearly suggests that NSCs and all of their derivatives (including neurons) are important in modifying the diseased cordal milieu via neurotrophic factor secretion. A subsequent study by the same group demonstrated that an even greater extension of survival and improvement in motor function could be achieved by transplanting hNSCs into both cervical and lumbar regions of the cord, despite greater surgical trauma (Xu et al., 2011). This proof of safety and efficacy is promising, given that multiple injections would be needed were this technique to be translated into human clinical therapy in order that cervical myotomes (controlling respiratory function) were influenced. The reality of this concept has since been substantiated in a meta-analysis of 11 standardized studies of murine and hNSC (lumbar and cervical) intraspinal injections in SOD1 transgenic mice. Studies in which hNSCs were transplanted into the cervical cord showed respiratory function preservation (and prolonged survival), attributed to the conservation of MNs and their functional connections, which was evinced by estimates of motor unit number (a motor unit comprises a single MN and the skeletal muscle fibres it innervates). These studies also demonstrated safety of the technique, as function and survival were not worse in any transplanted animal compared with controls, and neither tumour formation nor inappropriate cell proliferation were detected (Teng et al., 2012). Other groups have focussed their work on manipulating NSCs specifically to secrete growth factors in order to replace glia and support MN survival, although this work could also pave the way towards understanding endogenous repair and engineering cells to be able to stimulate it. Various studies have demonstrated the success of hNSC integration into the rodent spinal cord and subsequent functional growth factor secretion following both intraspinal (Klein et al., 2005) and intrathecal injection (Hwang et al., 2009). The latter, which demonstrated the migratory capacity of hNSCs (from the CSF space to their position of integration in cord) (Hwang et al., 2009), highlights the potential for alternative, less-invasive administration techniques, although these have not been rigorously compared. Early clinical trials demonstrate safety in injecting NSCs intraspinally Following successful pre-clinical studies of hNSC transplantation in ALS models, two in-human clinical trials are currently ongoing. The first Phase 1 trial was approved by the FDA in 2009 (National Library of Medicine at the National Institutes of Health), and aimed to assess the safety of the surgical procedure, hNSC implantation itself and the necessary immunosuppressive therapy. The trial, funded by Neuralstem, involved 15 patients and employed risk-escalation methodology: the first patients involved (who also received the fewest injections) were those with the most advanced disease, as less function stood to be lost were adverse events to occur. In the initial stages, six non-ambulatory and later six ambulatory patients received lumbar intraspinal injections of foetal cord-derived hNSCs and were followed up until 18 months. Safety was demonstrated in the first (interim) analysis, published in 2012 (Glass et al., 2012), by the absence of long-term complications resulting from either the hNSCs or the procedure itself, and the tolerance by all patients (Glass et al., 2012). It was agreed that the safety of cervical injections needed to be ascertained, as these would be imperative if the treatment were to have any impact on disease progression and lifespan. In addition, dual-targeted approaches (lumbar and cervical) offered further therapeutic potential through improving both limb motor and respiratory function. Consequently, three further patients, followed by three ambulatory patients who had received lumbar injections, received cervical intraspinal injections. Results published in 2014 reported that no patients experienced acceleration of disease progression and procedural tolerance was good (Feldman et al., 2014). Preliminary results of the second Phase I trial, conducted by the Mazzini group, further evidence the safety and tolerability of lumbar intraspinal hNSC transplantation (Mazzini et al., 2015). A Phase II trial to determine safety, feasibility, toxicity and maximum hNSC dose was started in 2013 (National Library of Medicine at the National Institutes of Health). The trials have also yielded promising results relating to clinical outcomes. In the Neuralstem trial's first stage, one patient exhibited clinical improvement, and in the second stage, over 50% patients demonstrated improvements in various clinical measures, and one showed slower than expected progression (Feldman et al., 2014). In the Mazzini trial, transient functional improvements were recorded in three patients (Mazzini et al., 2015). Whilst neither trial was sufficiently powered to determine treatment efficacy, making meaningful interpretation of these findings impossible, the promise demonstrated by both is encouraging. The rate at which in-human study is occurring is also exciting. Barriers and challenges to widespread clinical translation exist Although early clinical trials have demonstrated the safety of intraspinal hNSC injections in ALS, several issues need to be addressed before clinical translation is possible. Other factors may delay clinical application. Stem cells have long been a controversial area of research. Both clinical trials involving hNSCs obtained cells from foetal material (Feldman et al., 2014; Mazzini et al., 2015), which invites considerable ethical opposition. The Mazzini group suggested that they had overcome this issue by using brain tissue from naturally miscarried foetuses (Mazzini et al., 2015); however, this is unlikely to satisfy all groups. Complex regulatory issues accompany the ethical ones, and considerable international variability still exists regarding stem cell research regulations (Eurostemcell). As a consequence of using foetal (thus non-host) cells, graft survival relies upon indefinite immunosuppression. Tacrolimus and mycophenylate mofetil (immunosuppressive agents) were the major causes of toxicity in patients on the Neuralstem trial, and most patients required dose reduction or drug cessation as a result of significant gastrointestinal side effects; these effects are well known. Whilst no major opportunistic infections were clinically manifest, three patients developed fungal infections, and one developed a basal cell (skin) carcinoma (Glass et al., 2012). It is also worth considering the potential for immunosuppression in disease potentiation, given that inflammation contributes to ALS pathophysiology (Turner and Swash, 2015). A recent development in stem cell technology could provide a solution to the issues of ethics and immunosuppression. In 2006, a group in Kyoto developed a method of generating pluripotent stem cells from adult cells by introducing genes encoding four transcription factors (Takahashi and Yamanaka, 2006). These autologous cells, commonly derived from adult fibroblasts obtained by biopsy, can divide indefinitely and differentiate to form any cell type in the human body. As the technology is relatively new, induced pluripotent stem cells (iPSCs) have mainly been used to model disease in ALS so far (Coatti et al., 2015). However, a limited number of studies have investigated therapeutic applications. One such study, in which iPSC-derived NSCs were intrathecally and intravenously transplanted into ALS mouse models, demonstrated functional improvement and prolonged survival as a result of neurotrophic factor secretion and a reduction in microglial activity. These results provide evidence for both the efficacy and safety of iPSC-derived NSCs, as no abnormal cell proliferation and, importantly, no tumour growth were detected in transplant recipients (Nizzardo et al., 2014). Tumour formation is a risk that accompanies the use of all types of stem cell (Teng et al., 2012), and whilst they hold huge potential for future developments in cell replacement therapies, iPSCs have a greater propensity towards tumour formation than other stem cell types; the risk is significant (Lu and Zhao, 2013; Coatti et al., 2015). Whilst iPSCs could overcome ethical and immunosuppressive barriers to the clinical translation of NSC therapy, it would be counterproductive to replace one devastating disease with another. It is also worth noting that the challenge of standardizing transplant methodology may delay the therapeutic application of NSCs in ALS, as each method will require individual and comparative trialling (Lunn, Sakowski and Feldman, 2014). This applies to both cell derivation and surgical techniques, several of which have already been mentioned (Hwang et al., 2009, Nizzardo et al., 2014; Feldman et al., 2014). Conclusion The potential of NSCs to prolong survival and produce functional improvement in ALS has been widely evidenced in animal models. The results of early clinical trials are promising and demonstrate the safety of NSC transplantation in humans. Considerable progress has been made during the past decade, and so it is not unrealistic to expect NSC transplantation to reach clinical practice within the next 15 years, following successful completion of Phase III trials. Whether or not this could become routine is less certain, as many challenges still exist. Whilst NSCs can feasibly slow degeneration and provide neuroprotection, stimulating CNS regeneration is less realistic in the near future. To make this possible, further research is needed not only to understand endogenous repair mechanisms sufficiently to be able to stimulate them, but then also to enable full and safe control of regenerative processes. Unfortunately, NSCs are unlikely to constitute a cure for ALS. Author biography Amy is a medical student at King's College London in her final year of study. She recently completed an intercalated BSc in Neuroscience and Mental Health at Imperial College London with First Class Honours, and was awarded the Malcolm Morris Memorial Prize for best academic performance on the course. She has a keen interest in all areas of neuroscience and aspires to a career combining both clinical and academic neurology. Previous work has included developing a database for a regional Myasthenia Gravis service, and an audit of seizure presentations to a District General Hospital Emergency Department over a 2-year period. The latter was selected for a platform presentation at the Association of British Neurologists Annual Meeting in 2015, where Amy was awarded the Charles Symonds Prize for best audit. Amy was also the recipient of the 2015 Gowers’ Medical Student Award for an essay based upon this epilepsy audit. Outside of medicine, Amy is a member of the University of London Symphony Orchestra and holds DipABRSM with Distinction in viola performance. 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( 2006 ) Human neural stem cell grafts ameliorate motor neuron disease in SOD-1 transgenic rats , Transplantation , 82 ( 7 ), 865 – 875 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Supervisor: Dr Jane Saffell, Institute of Medical & Biomedical Education, St. George's University of London, Cranmer Terrace, London, SW17 0RE, UK. © The Author 2016. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com © The Author 2016. Published by Oxford University Press.

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Published: Jan 1, 2016

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