Scholarly article on topic 'The past, present and future of stem cell clinical trials for ALS'

The past, present and future of stem cell clinical trials for ALS Academic research paper on "Biological sciences"

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Experimental Neurology
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{ALS / "Amyotrophic lateral sclerosis" / "Stem cells" / "Clinical trials" / "Neurodegenerative disorder" / "Motor neuron disease" / Transplantation / "Stem cell therapy"}

Abstract of research paper on Biological sciences, author of scientific article — Gretchen M. Thomsen, Genevieve Gowing, Soshana Svendsen, Clive N. Svendsen

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the cortex, brainstem and spinal cord. This leads to paralysis, respiratory insufficiency and death within an average of 3 to 5years from disease onset. While the genetics of ALS are becoming more understood in familial cases, the mechanisms underlying disease pathology remain unclear and there are no effective treatment options. Without understanding what causes ALS it is difficult to design treatments. However, in recent years stem cell transplantation has emerged as a potential new therapy for ALS patients. While motor neuron replacement remains a focus of some studies trying to treat ALS with stem cells, there is more rationale for using stem cells as support cells for dying motor neurons as they are already connected to the muscle. This could be through reducing inflammation, releasing growth factors, and other potential less understood mechanisms. Prior to moving into patients, stringent pre-clinical studies are required that have at least some rationale and efficacy in animal models and good safety profiles. However, given our poor understanding of what causes ALS and whether stem cells may ameliorate symptoms, there should be a push to determine cell safety in pre-clinical models and then a quick translation to the clinic where patient trials will show if there is any efficacy. Here, we provide a critical review of current clinical trials using either mesenchymal or neural stem cells to treat ALS patients. Pre-clinical data leading to these trials, as well as those in development are also evaluated in terms of mechanisms of action, validity of conclusions and rationale for advancing stem cell treatment strategies for this devastating disorder.

Academic research paper on topic "The past, present and future of stem cell clinical trials for ALS"

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ELSEVIER

Review

The past, present and future of stem cell clinical trials for ALS

Gretchen M. Thomsen a,\ Genevieve Gowing a,\ Soshana Svendsen a, Clive N. Svendsen ^^

a Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA b Department of Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the cortex, brainstem and spinal cord. This leads to paralysis, respiratory insufficiency and death within an average of 3 to 5 years from disease onset. While the genetics of ALS are becoming more understood in familial cases, the mechanisms underlying disease pathology remain unclear and there are no effective treatment options. Without understanding what causes ALS it is difficult to design treatments. However, in recent years stem cell transplantation has emerged as a potential new therapy for ALS patients. While motor neuron replacement remains a focus of some studies trying to treat ALS with stem cells, there is more rationale for using stem cells as support cells for dying motor neurons as they are already connected to the muscle. This could be through reducing inflammation, releasing growth factors, and other potential less understood mechanisms. Prior to moving into patients, stringent pre-clinical studies are required that have at least some rationale and efficacy in animal models and good safety profiles. However, given our poor understanding of what causes ALS and whether stem cells may ameliorate symptoms, there should be a push to determine cell safety in pre-clinical models and then a quick translation to the clinic where patient trials will show if there is any efficacy. Here, we provide a critical review of current clinical trials using either mesenchymal or neural stem cells to treat ALS patients. Pre-clinical data leading to these trials, as well as those in development are also evaluated in terms of mechanisms of action, validity of conclusions and rationale for advancing stem cell treatment strategies for this devastating disorder.

© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/3.0/).

ARTICLE INFO

Article history: Received 9 December 2013 Revised 13 February 2014 Accepted 25 February 2014 Available online xxxx

Keywords: ALS

Amyotrophic lateral sclerosis Stem cells Clinical trials

Neurodegenerative disorder Motor neuron disease Transplantation Stem cell therapy

Contents

Introduction..................................................................................................................................0

Clinical trials ................................................................................................................................0

Mesenchymal/blood-derived stem cells for ALS..............................................................................................0

Intraparenchymal spinal cord delivery of MsC: The Mazzini Clinical Trials......................................................................0

Overview ........................................................................................................................0

Pre-clinical trials: Using MsC in proof-of-concept animal studies ......................................................................0

Intrathecal transplantation of MSCs induced to secrete neurotrophic factors (MSC-NTFs): BrainStorm Clinical Trial (NurOwn™) ....................0

Rationale and proof-of-concept studies..............................................................................................0

Overview ........................................................................................................................0

Martinez Clinical Trial: Frontal cortex transplantation of blood-derived stem cells..............................................................0

Mesenchymal stem cells for ALS: Additional considerations ..................................................................................0

Neural stem cells for ALS..................................................................................................................0

Spinal-derived neural stem cells for the treatment of ALS: Neuralstem Clinical Trial (NSI-566RSC)................................................0

Rationale and proof-of-concept studies..............................................................................................0

Overview ........................................................................................................................0

Glial restricted progenitor transplantation..................................................................................................0

Neural progenitor cells producing GDNF: An ex vivo gene therapy approach ..................................................................0

Vescovi Clinical Trial......................................................................................................................0

Discussion: Considerations for future clinical trials ..............................................................................................0

* Corresponding author at: Regenerative Medicine Institute, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA.

E-mail address: Clive.Svendsen@cshs.org (C.N. Svendsen). 1 Equal contribution.

http: //dx.doi.org/10.1016/j.expneurol.2014.02.021

0014-4886/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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Importance of pre-clinical studies..........................................................................................................0

Inclusion of placebo/sham patient groups ..................................................................................................0

Tracking transplanted cells................................................................................................................0

Clinical trial design strategies..............................................................................................................0

Biomarkers........................................................................................................................0

Route/location of administration ....................................................................................................0

Conclusions ..................................................................................................................................0

Acknowledgments ............................................................................................................................0

References....................................................................................................................................0

Introduction

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons in the cortex, brainstem and spinal cord resulting in paralysis and death within an average of 3 to 5 years from disease onset (Zinman and Cudkowicz, 2011). The majority of ALS cases are of unknown etiology and sporadic in nature (90-95%) with no genetic association. However, familial ALS (fALS) also exists and is associated with genes such as TAR DNA-binding protein 43 (TARDP, representing 2-6% of fALS cases), Cu/Zn superoxide dismutase 1 (SOD1, ~20%) and most recently discovered C9ORF72, representing an estimated 34% of all fALS cases (van Blitterswijk et al., 2012; Wijesekera and Leigh, 2009). Although understanding the mechanism and function of C9ORF72 and its role in fALS has recently come to the forefront of ALS research, previous studies have primarily relied on fALS cases caused by point mutations in the SOD1 gene. Toxic "gain of function" mutations in the SOD1 gene in transgenic rodents result in ALS-like phenotypes that have enabled researchers to study disease pathophysiology. While the SOD1G93A transgenic model and other rodent models of ALS have provided insight into disease mechanisms, the cause of motor neuron death in the more prevalent cases of sporadic ALS is still unknown. ALS patients experience upper limb, lower limb or bulbar onset, with variable involvement of upper and lower motor neurons and subsequently differing rates of disease progression (Kiernan et al., 2011; Ravits and La Spada, 2009). This heterogeneity of ALS makes it difficult to identify the mechanisms of disease origin and to develop successful therapies. Currently, ALS has no available pharmacological treatment options that offer long-term efficacy. After half a century of trials and testing of over 150 different therapeutic agents or strategies in preclinical models of ALS, Riluzole is the only developed drug that prolongs patient survival time, although by only approximately 2-3 months.

Human embroyonic-derived stem cells and induced pluripotent stem cells are perhaps the most powerful type of stem cell and hold great promise for the field. Recent advances in adult derived induced pluripotent stem cells avoids ethical concerns, and could allow for autologous cell transplantation in the future (Svendsen, 2013). Replacing lost motor neurons using human pluripotent stem cells is a potential therapy for ALS, but relies on transplanting motor neurons expected to survive and form long-distance projections (from brain to spinal cord, and/or spinal cord to muscle) with functional connections. This is a very challenging proposition given the long distances required for axo-nal outgrowth in the adult compared to development (when the motor neurons originally make contact with the muscle). In addition, the new motor neurons are placed in a very toxic environment where all those around them are dying. Alternatively, transplanting cells that have the ability to support the survival of existing motor neurons or potentially "detoxify" the environment is a far more practical idea. Support cell types can be generated from other tissues such as mesenchymal stem cells (MSCs) isolated from the bone marrow and neural stem cells (NSCs) isolated from fetal brain. These cells are not as likely to make primitive tumors as those derived from pluripotent cells. They are a safer potential product and have provided a much faster path to the clinic — although pluripotent stem cell research is developing rapidly and clearly this field will expand enormously in the coming years.

There have been several advancements using MSCs and NSCs, all of which rely on using the stem cells to stimulate the survival of existing motor neurons rather than motor neuron replacement itself. Mesenchy-mal and neural stem cells have been used to generate immunomodula-tory cells, growth factor-releasing cells, functional support cells such as glia, or GABAergic interneurons to modify motor neuron survival and activity (Gowing and Svendsen, 2011; Lunn et al., 2009; Maragakis, 2010; Papadeas and Maragakis, 2009). Here we discuss these therapeutic approaches and present a detailed synopsis of completed, current and future clinical trials that show the potential of mesenchymal and neural stem cells for the treatment of ALS (summarized in Table 1). Another recent review has also included many studies in the eastern academic press and provides an alternative view of current efforts to use stem cells to treat this devastating disorder (Meamar et al., 2013). Furthermore, for additional reference, "The ALSuntangled Group" has been instrumental in stimulating discussions and raising the awareness of certain clinical trials that have only been briefly mentioned here due to minimal pre-clinical data and/or arguable shortcomings in trial design and execution (The ALSuntangled group (2010)).

Clinical trials

Mesenchymal/blood-derived stem cells for ALS

MSCs are multipotent adult stromal stem cells of mesodermal lineage that can be easily isolated from various adult connective tissues including bone marrow and adipose tissue and subsequently expanded in vitro to provide large numbers of cells (de Girolamo et al., 2013; Deans and Moseley, 2000; Giordano et al., 2007; Laroni et al., 2013; Pittenger et al., 1999; Prockop, 1997). MSC are a very attractive candidate for cell therapy as they avoid the ethical and practical issues of embryonic and fetal-derived stem cells and provide the possibility of autologous transplantation. In most cases their mode of action for central nervous system (CNS) disorders is thought to be through transient effects on either inflammation or neuronal cell survival, partially through the release of growth factors and cytokines. There are few reports of robust MSC survival for more than a few weeks following transplantation, although this remains an area of debate (Castro et al., 2002; Himes et al., 2006; Li et al., 2001). A recent review of clinical trials using intravascular delivery of MSCs for various conditions concluded that MSC therapy appeared safe but that larger, well-controlled clinical trials with a rigorous reporting system were required to further confirm this safety (Lalu et al., 2012).

¡ntraparenchymal spinal cord delivery ofMsC: The Mazzini Clinical Trials

Overview

In 2003, Mazzini and colleagues performed some of the world's first clinical studies to determine the safety and tolerability of direct intraparenchymal transplantation of MSCs (or any cell type) to treat ALS. MSCs were isolated from allogeneic ALS patient bone marrow aspirates via a Percoll gradient centrifugation protocol and expanded in adherent cultures with medium containing fetal bovine serum. The authors stated that the final cellular product did not express hematopoietic markers CD45 and CD14, did express CD90, CD106, CD29, CD44,

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Table 1

Summary of past, ongoing and potential stem-cell based clinical trials for ALS.

Trial name

Treatment cell type Cell specifics

Delivery method

Trial status

Pre-clinical rationale for clinical translation

Mazzini Brainstorm

Martinez Neuralstem

Stem cells NSC

Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow

Autologous, derived from patient blood Human fetal spinal cord (at 8 weeks)

Spinal cord injections Intrathecal, intramuscular

Frontal cortex injections Spinal cord injections

Q Therapeutics GRP

Human fetal forebrain (at 17-24 weeks) Spinal cord injections

Cedars-Sinai hNPC releasing GDNF Human fetal cortex (at 8-15 weeks) Spinal cord injections

Vescovi NSC Human fetal (unspecified) Spinal cord injections

Phase I complete, no current studies Phase I complete, Phase lla current

Phase I complete, Phase II current Preclinical

Preclinical Phase I current

In vivo secretion of neurotrophic factors, beneficial effects in Parkinson's and Huntington's disease rodent models N/A

Enhanced survival of spinal motor neurons in rats

Beneficial effects on motor function, lifespan and spinal motor neurons, decreased microgliosis in rats; using rat GRPs

GDNF secretion in vivo enhances survival

of spinal motor neurons in rats

CD105, CD166 and retained the capacity to be differentiated into osteoblasts, chondroblasts and adipocytes, although this data was not shown in any detail (Ferrero et al., 2008; Mazzini et al., 2003; Mazzini et al., 2003; Mazzini et al., 2008). Remarkably, there was no published preclinical data supporting either efficacy or a mechanism of action for these cells following injection to the spinal cord of animals, although some animal studies were published following the clinical trial (see next section).

In an initial clinical study, seven ALS patients were transplanted with variable numbers of MSCs (7-152 x 106 cells) into the thoracic spinal cord with varying numbers of injection sites (2-5 sites). While there was no functional improvement over-time following MSC transplantation, no serious side-effects and no detrimental effects on neurological function were reported and the authors concluded that the transplantation of these cell suspensions into the human ALS spinal cord was safe and well tolerated Mazzini et al., 2003. In patient follow-up studies more than four years after surgery, no signs of toxicity or abnormal cell growth were detected and it was suggested that the treatment might have benefited four patients (Mazzini et al., 2006; Mazzini et al., 2008). However, Badayan and Cudkowicz report that the small sample size, disease variability in selected patients, lack of control groups, inconsistency in the cell number administered to the patients, and incomplete data presentation prevented a definitive conclusion on MSC safety and efficacy. Hence, they suggested that the lack of pre-clinical data supporting the approach should preclude further studies in ALS patients. (Badayan and Cudkowicz, 2008).

Though data from the initial clinical trial showed no overt functional effects, a second Phase I clinical trial was conducted with expanded patient numbers using the same methods as described in the original trial. Twenty patients were selected from 270 clinically definite and probable ALS patients, who were observed between September, 2003 and June, 2006. However, due to patient dropout and exclusions based on other criteria after the start of the study, the final cohort included only 10 patients (Mazzini et al., 2010). In a separate long-term safety study, a group of 19 ALS patients enrolled in two consecutive Phase I clinical trials between 2001 and 2003 and were followed for up to 9 years after surgery (Mazzini et al., 2012). From these patients, no correlation was found between the cell dose or number of injection sites and the severity and duration of side-effects. Importantly, neuroradiologic analysis demonstrated a lack of tumor formation and no abnormal cell growth. While ALS disease progression in the majority of patients did not appear to be slowed by the MSC transplant, it did not accelerate disease progression and the results from these studies show that this treatment is safe and does not have any major immediate or long-term harmful consequences.

Pre-clinical trials: Using MsC in proof-of-concept animal studies

Since the start of the Mazzini Clinical Trials, a number of studies in animal models of ALS have investigated the therapeutic potential of MSCs administered either peripherally or injected directly into the spinal cord. As MSC graft survival is seldom observed in affected CNS tissues following transplantation, the beneficial effects on disease pathology have often been attributed to transient immunomodulatory capacities or trophic factor production by grafted cells. However, in recent studies, bone marrow or adipose-derived murine MSCs were shown to survive in the CNS of SOD1G93A mice following intravenous administration (Marconi et al., 2013; Uccelli et al., 2012). The administration of bone marrow-derived MSCs to symptomatic ALS mice halted the decline in body weight, delayed the loss of motor function and extended lifespan by 17 days. Adipose-derived MSCs did not affect lifespan, but delayed the deterioration of motor performance and transiently increased the number of surviving motor neurons in ALS mice. Although reporterlabeled MSCs survived and appeared to have functional effects, the results could have been further strengthened by examining cell-type specific markers, performing histology on sham-treated controls to define background staining, and providing both low and high magnification images of grafted cells. Moreover, as MSCs administered into the circulation can often associate with blood vessel surfaces, using endothelial cell markers and confocal microscopy would discriminate the transplant location between CNS parenchyma and vasculature to assure that the cells indeed entered and survived in the CNS.

Using a different approach, the Mazzini group has now shown that human bone marrow-derived MSCs can be injected directly into the lumbar spinal cord of SOD1G93A transgenic mice (Vercelli et al., 2008). Though the title of this report implies that the MSCs had a robust effect in the ALS animal model, the data was less convincing. While stereolog-ical quantification revealed that transplantation of human MSCs resulted in a 1.5 and 2.5 fold increase in the number of cresyl violet-stained motor neurons in treated females and males, respectively, there were no significant benefit on functional motor behavior or survival in female transgenic mice. Furthermore, the delayed motor function decline and increased survival reported for MSC-treated male SOD1G93A mice was confounded as the study did not follow all animal groups to disease endpoint. A better powered study and a Kaplan-Meier survival curve would more accurately determine the benefit of this treatment on motor neuron survival and function (Scott et al., 2008). In addition, rather than labeling human cells with commonly used human-specific antibodies, a gold standard to detect human cells grafted into animals, cells were labeled with a dye (bisbenzimide) prior to transplantation. This dye and others have been shown to transfer from the labeled transplanted cells into host cells (Iwashita et al., 2000). Thus, better evaluation of

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grafted cell detection must occur to conclusively determine the presence of human MSCs in the mouse CNS in these studies.

intrathecal transplantation of MSCs induced to secrete neurotrophic factors (MSC-NTFs): BrainStorm Clinical Trial (NurOwn™)

Rationale and proof-of-concept studies

Several pre-clinical and clinical trials have used human bone marrow stromal-derived MSCs that are differentiated into specialized neuron-supporting cells to stably secrete neurotrophic factors (MSC-NTFs). These cells, from BrainStorm Cellular Therapeutics, have been trademarked as NurOwn™ since they can be used for autologous adult stem cell therapy. They have been shown to exhibit MSC markers, display spindle-like cell morphology, form single-cell-derived colonies, readily differentiate into adipocytes and osteoblasts, and have the capacity to express a number of neural genes. (Blondheim et al., 2006). Sadan and colleagues later described the differentiation of these human MSCs into cells that express the astrocyte markers glial fibrillary acidic protein (GFAP) and glutamine synthase, and that secrete brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and insulin-like growth factor-1 (Sadan et al., 2009). Beneficial effects were observed after intrastriatal transplantation of these cells into animal models of Parkinson's disease and Huntington's disease (Sadan et al., 2009; Sadan et al., 2012). In the 6-OHDA lesion model of Parkinson's disease, MSC transplants alone had no protective effect on pathology. In contrast, tyrosine hydroxylase-positive staining increased and rotational behavior decreased in animals treated with MSC-NTF compared to vehicle-treated controls. However, tissue analysis using a human-specific marker revealed very little survival 50 days following transplantation of the MSC-NTF cells. As MSC-NTFs were transplanted 24 h following injury and they do not seem to survive over time, these results indicate that the amelioration in pathology is likely due to the transient production of trophic factors. This raises the question of whether this treatment would be efficient in a chronic model of neurodegeneration. In the Huntington's disease studies, transplantation of MSC-NTF resulted in amelioration of apomorphine-induced rotation and increased striatum size compared to control. In contrast to their previous work, where transplanted cells were identified using human-specific markers, here the transplanted cells were labeled with the general membrane marker, PKH-26, for identification. While this method showed significant cell survival at 6 weeks following transplantation, no representative pictures of grafted cells were provided. To date, there are no peer-reviewed, proof-of-concept studies using these cells in an ALS animal model and therefore it is extremely difficult to assess the mechanism of action or discuss pre-clinical rationale for their use in the treatment of motor neuron degeneration. Nonetheless, the autologous transplantation of MSC-NTF in ALS patients is currently under investigation and described below.

Overview

In 2010, a Phase I/II open-safety clinical trial by Karussis and colleagues at the Hadassah Medical Center in Jerusalem, Israel showed that intrathecal and intravenous administration of autologous bone-marrow-derived MSCs into ALS patients is feasible and safe (Karussis et al., 2010). In this study, patients with ALS or multiple sclerosis were treated either via a standard lumbar puncture (~55 x 106, ~63 x 106 MSCs, respectively) or intravenously (~24 x 106 MSCs for both ALS and MS patients). While the definitive survival of injected cells was not shown, this treatment induced immediate immunomodulatory effects and was deemed safe. Although this study was not designed to detect therapeutic efficacy of this treatment, encouragingly, ALS patient ALSFRS scores remained stable for up to 6 months following treatment.

A more recent Phase I/II clinical trial by the Karussis group and BrainStorm Cellular Therapeutics evaluated the safety, tolerability and therapeutic effects of transplanting MSC-NTF cells (used in the Parkinson's and Huntington's disease animal studies described above)

into ALS patients. Twelve patients affected by early stage or progressive ALS were transplanted either intramuscularly or intrathecally with a single dose of MSC-NTF cells. This clinical trial spanning June 2011 to March 2013 is not yet published but details are found at (ClinicalTrials.gov NCT01777646). More recently, a Phase IIa dose-escalating trial has been designed to evaluate the safety and preliminary efficacy of MSC-NTF cell therapy at various doses in ALS patients. Beginning in 2013, twelve ALS patients in three cohorts were administered MSC-NTF in a combined therapeutic approach of both intramuscular and intrathecal injections with increasing doses. Detailed results of these ongoing clinical trials have not yet been published, thus no conclusion on the safety and efficacy of this approach can be made. While these studies are interesting, they are all open-label and therefore a well-controlled double blind Phase II clinical study is required to avoid observer bias and placebo effects. Additionally, the cell administration methods are different to the pre-clinical studies transplanting these cells in other disease models directly into the striatum. Therefore, it remains to be seen whether the chosen routes of administration are optimal for MSC-NTF to migrate, survive and exert maximal beneficial effects in the brain and spinal cord regions largely affected by ALS.

Martinez Clinical Trial: Frontal cortex transplantation of blood-derived stem cells

There has been one published trial from the Hospital San Jose Tecnologico de Monterrey in Mexico in which blood-derived stem cells were transplanted into the frontal cortex of ALS patients to target the upper motor neurons (Martinez et al., 2009). The rationale for this trial, however, was not clear. There was no pre-clinical animal data to show efficacy when similar blood-derived stem cells were transplanted into the cortex, questioning the progression of this technique to the clinic. There has been no evidence of efficacy of the procedure in patients (who were required to pay a minimum of $18,000 for participation in the trial) and there have been no reports of subsequent clinical trials using this technique. For further critical review of this study, see The ALSuntangled Group (2010).

Mesenchymal stem cells for ALS: Additional considerations

While MSCs are of great interest to the ALS community, the above studies are often associated with technical caveats and insufficient information supporting a delivery route and a rationale for their use in ALS. To our knowledge,there is no robust pre-clinical data in a relevant ALS disease model detailing the long-term safety, in vivo differentiation, dosing and biological activity of human MSCs proposed for use in patients.

However, MSCs do have several attributes that make them a promising candidate for cell-based therapies. First, they are easy to derive and provide an abundant source of cells. In addition, they are not immunogenic, they release growth factors and have immune-suppressive features (Aggarwal and Pittenger, 2005; Di Nicola et al., 2002; English and Wood, 2013; Le Blanc, 2003; Stagg and Galipeau, 2013). In fact, several clinical trials are evaluating the potential of systemic administration of MSCs for the treatment of inflammatory diseases such as graft-versus-host disease, multiple sclerosis and Crohn's disease (De Miguel et al., 2012; Figueroa et al., 2012). Together these promising attributes support the use of MSCs for cell therapy in ALS, which may also show inflammatory changes during progression. However, prior to further pursuit as a therapeutic approach for ALS, significant efforts should focus on demonstrating their therapeutic potential and putative mode of action in a well-powered and controlled pre-clinical study using a relevant ALS disease model. Even though this data is not available, MSC safety and ease of production have led to several current and completed Phase I clinical trials for determining the safety of intravenous, intraventricular or intrathecal delivery of MSCs to ALS patients (NCT01609283;

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NCT01142856; NCT01759784; NCT01759797; NCT01771640). While dramatic clinical improvement has yet to be seen in any of these trials, they will at least provide definite data about the safety of MSCs for ALS patients and they may lead to larger blinded trials to uncover any disease modifying effects of these cells.

Neural stem cells for ALS

ALS mouse studies have shown that motor neuron disease is a non-cell-autonomous process, as expression of mutant SOD1 within mouse motor neurons alone does not lead to an ALS phenotype (Jaarsma et al., 2008; Yamanaka et al., 2008a). Rather, non-neuronal cells such as glia also contribute to ALS pathology, which is demonstrated by the fact that deletion of the SOD1 mutation from microglia and astrocytes resulted in enhanced survival of transgenic ALS mice (Boillee et al., 2006; Clement et al., 2003 Yamanaka et al., 2008b). Healthy cells not expressing an ALS-associated mutation could also delay degeneration and significantly extend the lifespan of ALS mice. In addition to chimeric mouse studies, transplantation studies show that rodent glial cells transplanted into animal models of ALS have a beneficial effect on motor neuron survival and animal function (Lepore et al., 2008). However, in contrast to the positive effects from rodent-derived cells, grafting the equivalent human-derived cell type resulted in no functional benefits in ALS animal models (Suzuki etal., 2007; Lepore etal., 2011; Hefferan et al., 2012). This lack of functional effect of the human cells could be due to several factors: (i) Animal models have an extremely severe and rapid disease phenotype, (ii) the amount of cells (dose) or the number of transplant sites was insufficient to protect diseased motor neurons, (iii) human cell transplants into animals do not have time to fully develop into the most mature and therapeutic cell type before the diseased animal reaches the terminal stage or (iv) support cells alone will not reduce motor neuron death in ALS.

Cell-based therapeutic approaches to ALS and other neurodegenera-tive disorders have a long history with the use of primary fetal tissues and more recently NSCs (Lindvall, 1991). As replacing motor neurons in ALS is not currently practical, the focus instead is on providing support cells, which is validated by the above studies with rodent cells. There is also some evidence that providing new interneurons that connect with degenerating motor neurons may have some beneficial effects (Xu et al., 2006; Yan et al., 2006). This was the basis for one of the first clinical trials using stem cells for ALS as described below.

Spinal-derived neural stem cells for the treatment of ALS: Neuralstem Clinical Trial (NSI-566RSC)

Rationale and proof-of-concept studies

NSCs can be isolated from the CNS ofpost-mortem fetal samples and expanded in culture (Cattaneo and McKay, 1991; Gage et al., 1995; Svendsen et al., 1997; Wright et al., 2006). These cells have the capacity to become astrocytes, the most abundant cell type in the brain, as well as neurons and in some cases, oligodendrocytes. One such cell line, NS1-566RSC (Neuralstem, Inc.; Rockville, MD), was isolated from an 8-week-old human fetal spinal cord and expanded in culture as a monolayer using the FGF-2 mitogen, a method based on numerous rodent studies. Following transplantation into the rodent spinal cord, NS1-566RSC cells predominantly differentiated into neuronal cells expressing inhibitory (GABAergic) or excitatory (glutamatergic) cell fate markers, with some expression of astrocyte (GFAP) or oligodendrocyte (APC) markers (Guo et al., 2010; Yan et al., 2007). In early pre-clinical studies, intraparenchymal transplantation of NS1-566RSC cells into the lumbar spinal cord of SOD1G93A rat and mouse transgenic models of ALS enhanced motor neuron survival, delayed loss of motor function and extended lifespan by 11-12 days compared to control animals (Xu et al., 2006; Yan et al., 2006). In addition, transplantation of NSI-566RSC into both the lumbar and cervical spinal cord extended the survival of SOD1G93A rats by 17 days (Xu et al., 2011). Xu and colleagues

have also shown a significant increase in the expression of BDNF, GDNF and vascular endothelial growth factor in the spinal cords of animals grafted with NS1-566RSC, suggesting that motor neuron survival may be attributed to increased presence of trophic factors following transplantation (Xu et al., 2006).

Interestingly, more recent publications from the same group have shown that these differences in lifespan could be due to comparing treated animals with control animals that received dead cell transplants, which have been shown to exacerbate motor neuron death (Hefferan et al., 2012). In continuing studies that avoided the use of dead cells in the control group, NSI-566RSC cells transplanted into the spinal cord of ALS rats had no significant effect on animal motor behavior or lifespan (Hefferan et al., 2012). On the other hand, there was a significant increase in the number of spinal motor neurons at the sites of NSI-566RSC transplantation, suggesting that NSCs may in fact have a beneficial effect on ALS pathology. Therefore, the studies using NSI-566RSC cells in ALS animal models provide some support to translate this approach to a clinical setting.

In addition to rodent studies, the United States Food and Drug Administration (US FDA) often requires larger animal studies prior to clinical trials. As the pig spinal cord is similar in size to that of humans, this is a good large animal for modeling and optimizing surgical stem cell transplantation techniques. Validation of the surgical procedure and device necessary for the delivery of stem cells to the spinal cord was performed in pigs by Boulis and colleagues and shown to be safe (Federici etal., 2012; Raore et al., 2011; Riley et al., 2009; Riley etal., 2011).

Overview

Neuralstem, Inc. sponsored the first Phase I US FDA-approved clinical trial for a stem cell-based treatment of ALS. Initiated in 2010 and completed in 2013, this Phase I clinical trial involved the transplantation of human spinal cord-derived NSCs into the spinal cord of 15 late-to mid-stage ALS patients (Glass et al., 2012; Riley et al., 2012; Riley et al., 2014) (ClinicalTrials.gov identifier: NCT01348451; see Table 2 for overview of Neuralstem Clinical Trials). This initial trial was developed to assess the safety of the surgical technique and the implantation of human spinal cord-derived stem cells into ALS patients. This study had an "escalation of risk" design where lumbar microinjections were performed prior to attempts at cervical intervention and non-ambulatory patients received grafts prior to ambulatory patients.

In the first part of this study, 12 patients received either five unilateral or five bilateral (10 total) injections of human spinal cord-derived NSCs (NSI-566RSC line) into the lumbar region of the spinal cord (Glass et al., 2012) (for details see Table 2). There were no long-term surgical complications and importantly, ALS patients tolerated the procedure, gave no indications that the stem cells were injurious to the spinal cord and showed no disease acceleration due to injections. As transplantation of NSI-566RSC cells into the lumbar region appeared to be safe, the second part of the Phase 1 trial determined the safety of injecting cells into the C3-C5 cervical region of the spinal cord. Surgical intervention might pose a greater risk in this region that is responsible for respiratory and limb functions. Three new ALS patients and three patients that had previously received lumbar injections received unilateral cervical injections of NSI-566RSC cells. The primary outcome findings show that the vulnerable spinal cord of ALS patients is capable of tolerating up to 15 microinjections of these NSCs (Riley et al., 2014). A few negative events were observed following cell transplantation; however, it was uncertain that they were directly attributed to NSI-566RSC cells. Transient pain experienced by some patients was presumed to be associated with the injection procedure itself. Other observed adverse effects were attributed to ALS progression and the immunosuppressive regimen. There were some hints of positive effects (slowed rate of progression), particularly in patients with non-bulbar onset of disease, and one patient appeared to show an improvement in clinical status. However, one of the complications of small studies such as these is

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that certain rare patients may be mis-diagnosed or selectively responsive to the powerful immune-suppression regimes. Thus, these reports must be treated as anecdotal until confirmed in larger groups of patients.

The conclusion from this completed Phase I trial is that unilateral and bilateral transplantation of these human NSCs into the spinal cord of ALS patients is remarkably safe (given the invasive surgical techniques used) and very well-tolerated. It was also of note that there was no obvious deterioration in patient progression — an important possible outcome of infusions of cells into the fragile spinal cord of ALS patients. Based on these positive results, a Phase II study was initiated in September, 2013 to define a maximum tolerated dose of NS1-566RSC cells in ALS patients. Fifteen patients in five different dosing cohorts will receive advancing doses of up to a maximum of 40 injections and 400,000 cells per injection (Table 2). The first 12 patients will receive injections in the cervical spinal cord and the final three patients will receive both cervical and lumbar injections (Clinical trial identifier: NCT01730716).

Although the Neuralstem, Inc clinical studies are encouraging for ALS patients and the stem cell transplantation field, a number of challenges remain to be addressed. Most important of these are related to the management of adverse effects associated with chronic immune suppression of transplanted patients. This resulted in a number of patients in the Neuralstem trial being taken off their suppression due to severe side-effects. Interestingly, in two Parkinson's disease patients having received fetal midbrain transplants in the striatum or substantia nigra and receiving standard immunosuppression for a period of only 6 months, grafts were observed at post-mortem analysis 3-4 years following transplantation surgery (Mendez et al., 2005). This study seems to indicate that transient immune suppression may be sufficient to enable the survival of fetal grafts in the brain. Furthermore, in one notable study, patients who had received no immune suppression following fetal do-pamine neuron transplants showed surviving grafts up to 14 years after surgery (Freed et al., 2011). While primary fetal tissue may be very different immunogenically to expanded populations of NSCs, this study at least shows that the brain is an immunologically privileged site and more studies need to be performed to assess allograft survival in humans.

Glial restricted progenitor transplantation

Glial restricted progenitor (GRP) cells have the ability to adopt the fate of astrocytes or oligodendrocytes following differentiation in vitro or in vivo (Sandrock et al., 2010). In an initial study, GRPs were isolated from the rat embryonic spinal cord, sorted for the GRP-specific cell surface antigen A2B5, immortalized with the V-myc oncogene and engineered to express a reporter marker (Lepore et al., 2008). Following expansion in culture, GRPs were bilaterally transplanted into the cervical spinal cord parenchyma of the SOD1G93A rat model of ALS. At disease end-stage, there was robust survival of GRPs with differentiation into GFAP-expressing astrocytes in vivo. ALS rats grafted with GRPs showed a 1.5 fold increase in the number of motor neurons and a decrease in microgliosis compared to rats transplanted with dead cells. Additionally, ALS rats receiving GRP transplants exhibited a slower decline of grip strength, improved motor function score, a 16.9-day extension in lifespan and recording of compound action potentials in the phrenic nerve revealed better maintenance of diaphragm function compared to media and dead-cell transplanted control rats. A subsequent study assessed stem cell-derived astrocytes for the treatment of ALS by transplanting Q-Therapeutics' Q-Cells®, or human GRPs derived from the human fetal forebrain, into the cervical spinal cord of the SOD1G93A mouse model of ALS. Although grafted Q-Cells survived and predominantly differentiated into GFAP-expressing astrocytes in vivo, they had no effect on lifespan, motor behavior or motor neuron survival (Lepore et al., 2011).

The differing results from these two GRP transplantation studies may be due to the severity of the SOD1G93A mouse model of ALS compared to the less severe rat model, variation in the quantity of grafted cells, and several differences in cell type (rat versus human; spinal- versus forebrain-derived; allograft versus xenograft) including the slower rate of maturation of human cells compared to those derived from rodents. Despite the differences in results and the lack of a phenotypic effect, Q-Cells provided valuable insight into the fate of human GRPs transplanted into an ALS animal model. Additionally, no tumor formation or heterotopic engraftment was observed in the animals up to 3 months post-transplantation, an important finding for translation to the clinic. Due to the beneficial effects observed following the transplantation of the rodent GRPs and the apparent safety of the approach, preclinical studies are ongoing to permit QTherapeutics to file an Investiga-tional New Drug for clinical testing of Q-Cells for the treatment of patients with ALS.

Neural progenitor cells producing GDNF: An ex vivo gene therapy approach

In another approach, we have extensively studied human fetal cortex-derived neural cells that are cultured as 3-dimensional aggregates termed "neurospheres" in the presence of mitogens and passaged via a mechanical chopping method. At later passages, these cells differentiate in vitro into astrocytes and some neurons, but not oligoden-drocytes. Consequently, they are defined as bipotent neural progenitor cells (NPCs) rather than multipotent NSCs. Following transplantation into the CNS, cells preferentially differentiate into GFAP-expressing astrocytes, although neurons can arise when younger passages are transplanted (Fricker et al., 1999; Ostenfeld et al., 2000; Svendsen et al., 1997). However, transplantation of these human NPCs (hNPCs) into the lumbar spinal cord of the SOD1G93A rat model did not lead to phenotypic functional benefit or enhanced motor neuron survival (Klein et al., 2005; Suzuki et al., 2007). Conversely, cervical transplantation of these cells into the same ALS model specifically promoted the survival and the function of phrenic motor neurons ipsilateral to the transplant (Nichols et al., 2013).

Many stem cell transplantation studies involving animal models of CNS disease have attributed observed beneficial effects to grafted cells producing trophic factors, immunomodulatory molecules or other neu-roprotective factors (Gowing and Svendsen, 2011). We have previously shown that hNPCs can be genetically engineered to stably secrete neu-rotrophic factors in vivo (Fig. 1) (Ebert et al., 2008; Ostenfeld et al., 2000). In the context of motor neuron disease, GDNF initially attracted interest in 1994 because of its potent capability to enhance the survival of motor neurons in vitro and in vivo (Henderson et al., 1994). Unilateral transplantation of hNPCs secreting GDNF in the lumbar spinal cord of SOD1G93A transgenic rats resulted in a 3-fold greater number of surviving motor neurons ipsilateral to the graft compared to animals transplanted with control, non-GDNF-secreting hNPCs (Suzuki et al., 2007). Despite this significant increase in motor neuron survival, there was no functional benefit of GDNF-producing hNPCs on motor behavior or neuromuscular innervation. Interestingly, similar to the results obtained with hNPCs alone, unilateral transplantation of hNPCs expressing GDNF into the cervical spinal cord of ALS rats resulted in improved phrenic motor output at baseline compared to the contralateral non-transplanted side (Nichols et al., 2013). Recently, we have shown that hNPCs engineered to express GDNF survive long-term (7.5 months) following transplantation into the spinal cord of athymic nude rats and continue to secrete GDNF (Gowing et al., 2013).

Based on the results from these animal studies, hNPCs expressing GDNF are being pursued for clinical translation. A master cell bank of hNPCs generated under good manufacturing practice (GMP) has been produced and the expansion of clinical-grade lots of hNPCs transduced with a GMP-grade lentivirus encoding GDNF is currently ongoing. Prior to filing for an Investigational New Drug, four steps remain: (i) determination of the optimal effective and maximum feasible dose

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Neuralstem clinical trial patient summary. Phase I

Each injection administered to patients contained approximately 100,000 cells in an 8.5-10 |jL volume.

Group Spinal cord region Disease condition Total # of injections

A1 Unilateral/lumbar (L2-L4) Nonambulatory 5

A2 Bilateral/lumbar (L2-L4) Nonambulatory 10

B Unilateral/lumbar (L2-L4) Ambulatory 5

C Bilateral/ lumbar (L2-L4) Ambulatory 10

D Unilateral/cervical (C3-C5) Ambulatory 5

E* Unilateral/cervical (C3-C5) Ambulatory 5 * Patients in Group E were the same patients as in Group C, receiving additional injections at a later time point, for a total # of 15 injections.

Phase II

All patients in Phase II are ambulatory, early-stage with arm weakness, but not paralysis, receiving bilateral injections of varying cell doses suspended in an 8.5-10 |jL volume.

Group Spinal cord region Total # of cells Total # of injections

A Cervical C3-C4 2,000,000 10 (200,000 cells/injection)

B Cervical C3-C5 4,000,000 20 (200,000 cells/injection)

C Cervical C3-C5 6,000,000 20 (300,000 cells/injection)

D Cervical C3-C5 8,000,000 20 (400,000 cells/injection)

E Lumbar L2-L5, followed 4-12 weeks later with cervical C3-C5 160,000,000 40 (400,000 cells/injection)

in an ALS animal model, (ii) establishment of safety/toxicity, (iii) tumor-igenicity testing in immune-compromised rats and (iv) determination of the safety of cells and surgical approach in Yucatan mini-pigs as a pre-clinical model. The final goal is FDA approval of a Phase I/IIa trial with 18 patients receiving unilateral lumbar hNPC-GDNF transplants and systematic clinical assessment over a 12-month period.

Vescovi Clinical Trial

There is also one small trial currently being conducted by the Vescovi group from Azienda Ospedaliera Santa Maria, using NSCs derived from human fetal tissues (Gelati et al., 2013) for injection into the lumbar spinal cord of ALS patients in Italy. We could not find any pre-clinical published data to support this trial, however, it is listed on Clincialtrials.gov (ClinicalTrials.gov Identifier: NCT01640067).

Discussion: Considerations for future clinical trials

Importance of pre-clinical studies

Stem cell-based therapies come with multifaceted considerations regarding safety, cell availability, immune rejection and ethics in the case of embryonic- or fetal-derived cells (Aboody et al., 2011; Hyun et al., 2008; Lindvall and Kokaia, 2010; Lindvall et al., 2012). However, in devastating diseases such as ALS, where there is no prevention or effective treatment, clinicians and patients are likely willing to take more risks in their treatment options. Nonetheless, prior to clinical translation for ALS, scientific evidence must support the ability of the proposed treatment to either replace degenerating motor neurons or promote their survival and function (Lindvall et al., 2012). Unfortunately, therapeutic strategies that showed efficacy and promise in ALS animal models were less efficient in clinical trials. While this dichotomy suggests little value of proof-of-concept pre-clinical studies, it is also true that studies in animal models are often insufficiently powered, poorly controlled and rarely reproduced in identical or additional disease-specific models prior to pursuing clinical translation (Schnabel, 2008; Scott etal., 2008).

To identify therapeutic strategies pertinent to the human condition, it is essential to have rigorous and appropriately designed translational studies that have strong rationales and that consider the delivery method, treatment timing, critical controls, and relevant functional outcome. For example, recent trials using minocycline highlight the importance of incorporating multiple time points in pre-clinical studies. Three

independent studies showed minocycline treatment before the onset of overt ALS symptoms slowed disease progression in mutant SOD1 transgenic mice (Kriz et al., 2002; Van Den Bosch et al., 2002; Zhu et al., 2002). However, a randomized placebo-controlled Phase III trial conversely revealed that minocycline administration accelerated the decline of ALS patients (Gordon et al., 2007). This reversed effect of minocycline given to post-symptomatic patients was subsequently validated when ALS mice treated with minocycline after the onset of symptoms showed no increase in survival (Keller et al., 2011). Clearly, multiple time points along with many other aspects need to be carefully considered when using animal models for translation to clinical studies.

Inclusion of placebo/sham patient groups

There have been many discussions regarding the use of placebo patient groups in clinical trials involving complex and risky surgical procedures. Obviously this would increase confidence in the outcome being related to the implanted cells rather than simply to the injected vehicle. However, putting patients through such high-risk surgical procedures without possible benefit may not be acceptable to some Institutional Review Board committees. The Neuralstem Inc. trial did not have a placebo group, nor have any of the other transplant trials for ALS discussed in this review as far as we can establish. Interestingly, the use oftargeted delivery of stem cells to restricted regions of the CNS allows for more creative clinical trial designs, which both increase the power of the study and bring in a placebo control. In our own planned trial, GDNF-secreting stem cells will be transplanted only unilaterally into the lumbar spinal cord of ALS patients. While the neurosurgeons will know the transplanted side, the neurologists and patients will be blinded. This will allow the progression in both legs to be monitored over-time to establish whether the cell transplant had any effect on the treated side, without confounds from the patient knowing which leg may be expected to improve. While this is not a full placebo controlled trial (for this we would need to inject vehicle into the non-treated side) it does allow a blinded trial to occur through localized unilateral delivery of the cells. Typically in ALS, slow or fast paralysis progression in one leg predicts a similar paralysis progression in the other leg. This allows for a strong prediction ofleg paralysis progression in the same patient and, importantly, a determination of whether the leg paralysis progression is affected by unilateral cell transplants. This increases the power of the trial significantly when compared to trials involving many patients with very different progression rates.

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Fig. 1. Representative images of GDNF-expressing hNPCs transplanted in the rat spinal cord using human-specific antibodies. (A-C) Immunostaining for a human-specific cytoplasmic marker (red; STEM121, Stem Cells Inc.) and GDNF (green; R&D) at (A) 10x and (B) 40x. Notice the presence of GDNF in neuronal cell bodies (white arrows) and (C) lack of staining in the ventral horn contralateral to transplant. (D) Human nuclei stain (red; Ku80 antigen, STEM101, Stem Cells Inc.) and labeling for the motor neuron marker choline acetyltransferase (green; ChAT; AB144P, Millipore) showing the localization of the transplants in the spinal cord ventral horn. (E) Human-specific nestin (red; ABD69, Millipore) and human-specific GFAP (STEM123 Stem Cells Inc.) showing the in vivo phenotype of the grafted cells.

Tracking transplanted cells

A key challenge for stem cell-based therapeutic approaches is the need for reliable tracking of grafted cells. The ability to localize transplanted cells using imaging technologies would benefit the assessment of targeting and safety of the cells. Currently, radiolabelling cells with flurodeoxyglucose, indium or technetium and subsequent PET or SPECT imaging appears to be the most commonly used clinical approach (McColgan et al., 2011). However, the short half-life of the clinically approved molecules limits the capacity for long-term imaging. Moreover, the poor spatial resolution of acquisition techniques combined with the possible deleterious effects of ionizing compounds results in low appeal of these methods for CNS transplantation (McColgan et al., 2011). Another approach is iron-oxide-labeling of cells for magnetic resonance imaging (MRI). Although offering high resolution, the sensitivity of this modality is low. The most commonly used compounds are superparamagnetic iron oxide (SPIO) or ultrasmall SPIO nanoparticles. Some studies have shown encouraging data for tracking of NSCs following intravenous or intraparenchymal injection (Deng et al., 2013; Gutova et al., 2013), but our unpublished data (Bernau et al, manuscript in progress) and that of others have suggested that the T2-weighted MR signal or detection of iron deposits via Prussian blue stain, is not necessarily indicative of the presence of live cells (Sadan et al., 2009). Interestingly, in one study, the transplantation of cells labeled with bimodal MRI contrast agent, a Gadolinium-RhodamIne Dextran conjugate, abolished behavioral improvements and the reduction in lesion size compared to control rodents receiving unlabeled NSCs (Modo et al., 2009). Clearly, having the ability to use imaging modalities in the clinic to track cells following transplantation would be a significant advancement to the field. However, when considering labeling strategies, prior to moving to the clinic, it is important to consider the overall effect of

the label on cell viability both in vitro and in vivo, the possibility of any CNS toxicity and the specificity of the label for identifying live (versus dead) cells.

Clinical trial design strategies Biomarkers

Another important consideration for stem cell-based therapies is the overall clinical trial design. Biomarkers can indicate the biological effects of grafted cells on disease manifestation and can help predict the outcome on motor function during the patient's lifespan essential for indicating the biological effects of grafted cells on disease processes as well as for assisting in determining the outcome on patient motor function (Bruijn and Cudkowicz, 2014; Turner et al., 2013). Various tissues and fluids can be used to identify biomarkers including cerebral spinal fluid (CSF), blood, urine, saliva, muscle and skin (Turner et al., 2013). Currently the most promising biomarkers are found in the CSF and include those that are indicative of neuronal loss or neuroinflammatory processes (Ganesalingam et al., 2011; Puentes et al., 2014; Sussmuth et al., 2008). Measures of motor function such as motor unit number estimation, motor unit number index, and electrical impedance myography may also be valuable for potentially monitoring disease progression and therapeutic effects (Bromberg, 2013; Furtula et al., 2013; Nandedkar et al., 2004; Rutkove et al., 2014). However the majority of these markers have yet to be fully validated for routine use in ALS Clinical Trials.

Route/location of administration

The route of cell administration to patients is clearly of vital importance. There is little published evidence that cells delivered via any route other than direct parenchymal injection can get deep into the

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Stem Cell and Gene Therapy for ALS

Summary of Clinical Trails

Martinez/Hospital San Jose Tecnologico de Monterrev, Mexico (Intraparenchymal blood-derived stem cells)

si i\ \i ( ".ни Cervical Neuralstem/Emory (Intraparenchymal hNPCs) Q Therapeutics (Intraparenchymal GRPs)

Mazzini/Eastern Piedmont University, Italy (Intraparenchymal autologous MSCs)

Thoracic

tà/ Neuralstem/Emory (Intraparenchymal hNPCs) Cedars-Sinai (Intraparenchymal hNPC-GDNF) Brainstorm (Intrathecal autologous MSCs)

... 'Л Brainstorm ' \ (Intramuscular autologous MSCs) Skeletal Mlicii

*Completed/Ongoing *ln pre-clinical development

Fig. 2. Summary of clinical trials involving stem cells and gene therapy for the treatment of ALS. Schematic shows the regions affected by ALS including the brain, spinal cord and skeletal muscle and highlights the current and developing therapies to target these regions.

spinal cord around the dying motor neurons in animal models of ALS. Thus the most reliable technique remains direct injection of the cells into the spinal cord.

Interestingly, there have been no well-designed treatment strategies targeting the upper motor neurons in ALS patients. While little is known about brain pathology in ALS, a number of studies in both animal models and in patients propose that hyperexcitability and/or corticospinal motor neuron degeneration observed presymptomatically may be the initial trigger to disease onset (Ozdinler et al., 2011; Vucic et al., 2008). While many of the current stem cell-based therapies target spinal motor neurons, therapeutic benefit may also result from targeting corticospinal motor neurons using cell therapy and growth factors. It is also likely that a combined approach, in which multiple pathways and regions within the CNS/skeletal muscle are targeted simultaneously or in a well-designed time course, will be most successful in treating the disease. For instance, while transplanted neural progenitors secreting GDNF can promote the survival of spinal motor neurons, functional outcome is not altered in this model due to downstream disjunction of muscle endplates that have not been targeted and therefore not spared (Suzuki et al., 2007). In a different approach, we have shown that MSC transplants in the

muscle of ALS rats resulted in a mild amelioration of motor neuron function. This effect was enhanced following the transplantation of MSCs engineered to produce GDNF, which also resulted in an increased lifespan of the treated ALS rats (Krakora et al., 2013; Suzuki et al., 2008). Thus, it is possible that combining these various therapeutic approaches would lead to increased beneficial effects (Fig. 2).

Conclusions

Prior to initiating future clinical trials, proof-of-concept data must show that the stem cells can be targeted to the therapeutic area without any adverse effects. Pre-clinical and clinical trials with both MSCs and NSCs indeed suggest that novel stem cell therapeutic approaches are safe and feasible. Some stem cell approaches in animal models provide a strong rationale and evidence to suggest that stem cell therapy has great potential to delay motor neuron degeneration and potentially enhance function in ALS patients. While not all pre-clinical animal models support this hypothesis, it should be considered that these models are subject to many limitations such that final proof may require confirmation in humans affected by the disease. Indeed, based on the current safety of new stem cell transplantation approaches for ALS, this review highlights that it is now time to move promising stem cell-based therapies into the clinic in order to really understand whether they may work in patients.

Acknowledgments

We thank Nicholas Wede for graphic art. This work was supported by CIRM (CIRM DR2A-05320) and ALS Association (ID# LQLWE0).

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