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Stem cells for ALS: An overview of possible therapeutic approaches
Accepted Manuscript Title: Stem cells for ALS: an overview of possible therapeutic approaches Author: Joanna Czarzasta Aleksandra Habich Tomasz Siwek Adam Czapli´nski Wojciech Maksymowicz Joanna Wojtkiewicz PII: DOI: Reference:
S0736-5748(16)30037-5 http://dx.doi.org/doi:10.1016/j.ijdevneu.2017.01.003 DN 2151
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
23-2-2016 6-1-2017 6-1-2017
Please cite this article as: Czarzasta, Joanna, Habich, Aleksandra, Siwek, Tomasz, Czapli´nski, Adam, Maksymowicz, Wojciech, Wojtkiewicz, Joanna, Stem cells for ALS: an overview of possible therapeutic approaches.International Journal of Developmental Neuroscience http://dx.doi.org/10.1016/j.ijdevneu.2017.01.003
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Stem cells for ALS: an overview of possible therapeutic approaches Joanna Czarzasta1*, Aleksandra Habich2, Tomasz Siwek2, Adam Czapliński2,3, Wojciech Maksymowicz2, Joanna Wojtkiewicz1, 4, 5
1
Department of Pathophysiology, 2Department of Neurology and Neurosurgery, Faculty
of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland 3
Neurocentrum Bellevue, Neurology, Zurich, Switzerland
4
Laboratory of Regenerative Medicine, University of Warmia and Mazury, Olsztyn, Poland
5
Foundation for nerve cells regeneration, Olsztyn, Poland
* Corresponding author: Tel: 0048(89)5246133; fax: 0048(89)5277004 E-mail address: [email protected] (J. Czarzasta)
Department of Pathophysiology, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland
Highlights:
1, Amyotrophic lateral sclerosis (ALS) is an unusual, fatal, neurodegenerative disorder
leading to the loss of motor neurons.
2. Multiple factors are contributed to the progression of ALS.
3. Stem cells are the most promising tool used in the preclinical and clinical trials for ALS. In this review we would like to discuss therapeutic properties of this cells and introduce completed and ongoing clinical trials about stem cell therapy for ALS.
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Abstract Amyotrophic lateral sclerosis (ALS) is an unusual, fatal, neurodegenerative disorder leading to the loss of motor neurons. After diagnosis, the average lifespan ranges from 3 to 5 years, and death usually results from respiratory failure. Although the pathogenesis of ALS remains unclear, multiple factors are thought to contribute to the progression of ALS, such as network interactions between genes, environmental exposure, impaired molecular pathways and many others. The neuroprotective properties of neural stem cells (NSCs) and the paracrine signaling of mesenchymal stem cells (MSCs) have been examined in multiple pre-clinical trials of ALS with promising results. The data from these initial trials indicate a reduction in the rate of disease progression. The mechanism through which stem cells achieve this reduction is of major interest. Here, we review the to-date pre-clinical and clinical therapeutic approaches employing stem cells, and discuss the most promising ones.
Key words: amyotrophic lateral sclerosis, risk factors, stem cells therapy,
1. Introduction Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive neurodegenerative disorder affecting upper and lower motor neurons (MNs) in the cerebral cortex, brainstem and spinal cord [1]. Hence, the signs of damage to MNs are both at the peripheral (e.g. atrophy) and central (e.g. spasticity) level. To date, there has been no effective treatment for ALS; however, there is one US Food and Drug Administration approved treatment - Riluzole, which has a protective effect on motor neuron degeneration
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through excitotoxicity by blocking the release of glutamate, with has an impact on NMDA or AMPA receptors. This neuroprotective agent specifically inhibits sodium channels and modulates the GABAergic system, which indicates that it affects many pathogenic mechanisms [2]. The drug may extend patient survival by a few months, though only if used in the early stages of the disease [3]. The majority of patients die within 3 to 5 years after diagnosis, usually due to respiratory failure, while some may survive even up to ten years. Prevalence ranges from 1-2 to 4-6 per 100,000 individuals per year, and thus ALS is classified as a rare disease. New theories consider it to be a multi-systemic illness, rather than a disorder involving only motor neurons. Approximately 10% of patients suffering from ALS (motor symptoms predominate) exhibited symptoms of frontotemporal dementia (FTD) [4]. ALS is a very individual disorder with many factors leading to its onset. Some of them, so-called “predictive factors”, concern mainly age, sex and family history. The average age of incidence for ALS is 55-60 years, with the disorder affecting men more often than women [5]. Most cases of ALS are sporadic (sALS) with no family history, while only 10-15% of cases are familial (fALS), with dominant inheritance [6]. The etiopathogenesis and mechanisms that underlie ALS are not fully understood, but many risk factors have been considered, including genetic, environmental and epigenetic agents, as well as substantial changes in the motor neuron microenvironment [7]. Due to the modest effect of the only available drug in the treatment of ALS, the development of new and effective strategies has high priority and a variety of approaches are in various stages of development and clinical trials. Possible treatments are associated with anti-glutamatergic, anti-oxidant, mitochondrial, and antiinflammatory agents [8]. In order to deliver support to all of these trophic factors, gene therapy is also carried out. In recent years, stem cell therapy in ALS treatment has gained a lot of attention because of the multifaceted background of this disorder. The goal of this review is
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to: (1) introduce the existing risk factors involved in amyotrophic lateral sclerosis, and (2) discuss potential therapeutic stem cell strategies. 2. Suggested mechanisms responsible for ALS onset In the last two decades, several possible mechanisms and agents related to the origin, onset and development of ALS have been investigated. Despite the fact that the majority of ALS forms are sporadic, a wide range of genetic mutations have been identified in the autosomal dominant familial type of this disease [9]. In 20% of fALS patients, the most frequent mutation is linked to the gene encoding Cu/Zn superoxide dismutase 1 (SOD1) [5]. This mutation is connected with an antioxidant defense system against reactive oxygen species (ROS), which consist of three isoforms of SOD, including cytoplasmic Cu/ZnSOD (SOD1). The antioxidant defense system plays a critical role in inhibiting oxidative inactivation of nitric oxide. Indisputable evidence shows that oxidative stress is one of the mechanisms leading to the MN degeneration by an increase in markers of oxidative damage in the spinal cord and cerebrospinal fluid (CSF) of ALS patients [10]. Although oxidative damage in the pathogenesis of ALS is extensive, the precise mechanism(s) that results in elevated levels of ROS have not been fully explained. Other common genetic abnormalities causing both sALS and fALS include those involving the 43-kDa TAR DNA-binding protein (TARDP) gene, chromosome 9 open reading frame 72 (C9ORF72) gene and several others [5]. However, so far no ALS gene has been exclusively linked to only the ALS motor phenotype, indicating that ALS is a multi-system neurodegenerative disease with a propensity to affect the motor system. Apart from previous findings concerning the existence of the genetic predisposition to ALS, there are also environmental, so-called “non-lifestyle factors”, and lifestyle factors associated with the pathogenesis of this disorder. Among the factors not connected to lifestyle are neurotropic viruses and cyanobacterial toxins, magnetic fields, the metalloid selenium, and heavy metals as well as certain chemicals [11]. One of the examined
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heavy metals in ALS patients is lead, which was found to be in higher levels in several different tissues of people suffering from ALS [12-15]. It was also shown that work and home exposure to some chemicals, such as pesticides and fertilizers, appears to be greater in ALS cases as compared to control groups matched for age and gender [16]. In addition, the increased risk of ALS is associated with lifestyle factors, such as smoking, diet, history of trauma and intense physical activity [17]. The relationship between the genetic predisposition and environmental influence indicates the involvement of epigenetic factors in the pathogenesis of ALS. Epigenetic changes refer mainly to covalent post-transcriptional modifications of DNA, histone and chromatin remodeling, RNA editing, and non-coding RNAs. In consequence, the abovementioned alterations may regulate the gene expression and function [18]. All of these mechanisms manage neuronal development, plasticity, as well as aging, and are very important in maintaining cellular homeostasis [19]. ALS is not only related to genetic, environmental and epigenetic factors, but also to the changes in the microenvironment of motor neurons. Multiple events may contribute to the pathogenesis of ALS, such as excitotoxicity, oxidative stress, mitochondrial dysfunction, protein aggregation, dysregulation of RNA processing, neuro-inflammation, disruption of axonal transport and glial cell dysfunction [5, 20-21]. Of the above-mentioned events, neuroinflammation is a major player in the course of ALS, and is characterized by astrocyte and microglial activation, T cell infiltration, and the overproduction of proinflammatory cytokines and other factors. Astrocyte activation during the neuroinflammatory process leads to the secretion of cytokines and growth factors. It is known that astrocyte-derived transforming growth factor/TGF/-β1 fulfills an anti-inflammatory role in the neuroprotective inflammatory reaction in transgenic mice by regulating microglial activation, T-cell numbers, and interferon /IFN/-γ and interleukin /IL/-4 balance [22]. What is more, microglial cells, as
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primary mediators of neuroinflammation, may be transformed into phagocytes upon damage to the cell in the resting phase, or release inflammatory molecules in the active phase. Microglia may be activated in the early stages of ALS, or in themselves triggers the disease and plays a dual function, either protective or harmful. T lymphocytes, on the other hand, by involvement in the inflammation, have a contrarily protective function at early stages of ALS, though this effect is not sustained. They are also linked with microglial functions by inhibiting the harmful impact of microglia or enhancing the neuroprotective influence of these cells. Among the many components/molecules engaged in the inflammatory response, cytokines such as IL-1β, IL-7, IL-9, IL-12, IL-17, tumor necrosis factor-α and IFN-γ, as well as also chemokines were upregulated in CSF of ALS patients [23]. Similarly to microglia, cytokines/chemokines may exert a harmful or protective effect. Correlating their level with clinical ALS status, some of them may indicate worsened neurological function, while others may slow down the progression of the disease by improving the functional performance of the patient. As it had been mentioned above, ALS is a multi-factorial and multi-systemic disease, the effects of which include a reduction of blood-brain and blood-spinal cord barrier (BBB/BSCB) integrity. Under physiological conditions, BBB and BSCB are responsible for the control of brain homeostasis, regulation of influx and efflux transport, and protection from damage by their own specialized multicellular structure, such as the capillary endothelium, cell-tight endothelial junctions, capillary basement membranes and astrocytes [24]. Studies carried out on SOD1 mice and ALS patients showed that BBB and BSCB integrity is disrupted by damage to endothelial cell capillaries, astrocyte end-feet, perivascular edema, alterations to basement membrane components, etc. [24-25]. It seems that the role of decreased BBB and BSCB integrity in ALS pathogenesis still needs to be explained; however, there is evidence to suggest that, in the mice model, BSCB disruption occurs at an early stage of the disease and that restoring barrier integrity delays the disease process [26].
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Additionally, not only neuroinflammation but also the immune response may play a crucial role in the pathological progression of ALS [27]. During the onset of symptoms and course of the disease, alterations in the structure, quantity and role of immune cells have been found outside and within the central nervous system (CNS) [28]. It has been shown that peripheral blood lymphocytes of ALS patients have a different functionality, presumably because of differences in the immune response depending on the stage of the disease. Extensive lymphocyte proliferation in ALS patients with mitogen induction may be the result of autoimmunity alterations, while non-inducible proliferation in such patients may point to an immune deficiency [29]. During ALS progression, changes occur not only in lymphocyte proliferation, but also in the systemic pro-inflammatory state and in the antioxidant system. Higher concentrations of pro-inflammatory IL-6, IL-8 and nitrite, as well as reduced antioxidant glutathione levels, might suggest adaptive immune responses dependent on the stage of disease in the effected individuals [30]. 3. Preclinical advances in stem cell therapies for ALS In spite of the many years of trials and tests seeking an effective drug or therapeutic strategy, a method of controlling or inhibiting ALS has not yet been established. As it was previously mentioned, Riluzole is the only approved drug, prolonging patient survival for only a very short period. The future of preclinical and clinical trials is particularly linked to genetic engineering and stem cell therapy. In this review, we focus solely on the available stem cell types and their application in ALS treatment. The main distinguishing features of individual cell populations are their source of origin, accessibility, and potential for differentiation [31]. Due to their unique features, stem cells are an extremely attractive tool in ALS therapy as they may offer neuro-protection by exerting a paracrine effect and replacing degenerated neural cells [32]. 3.1.Neural Stem Cells
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Neural Stem Cells (NSCs) represent a population of progenitor cells located in the developing and mature CNS specifically in neurogenic areas of the brain, such as the subventricular and sub-granular zone [33-34]. The neurogenic niche provides a special microenvironment that determines the lifespan and fate of NSCs. This is related to the paracrine effect of these cells, including cell–cell contact and interaction with specific components of the extracellular matrix. Also, these cells in an undifferentiated form may exert “bystander” action in regard to cell replacement in inflammatory CNS disorders. Such an effect occurs when NSCs provide neurotrophic support in response to stimuli elicited by T cells within CNS, at the level of atypical perivascular areas of the multiple sclerosis (MS) animal model [35]. It needs to be highlighted that NSCs secrete extracellular vesicles (EVs) which communicate with the host’s immune system and mediate a neuroprotective and immunomodulatory effect via release their components (lipids, proteins and nucleic acids) [36]. If EVs pass the BBB and get into nerve cells during the inflammatory process, they could heal these cells; however, such cellular and molecular mechanisms have not yet been fully elucidated. NSCs are characterized by the high potential of self-renewal, properties of multipotency, i.e., the ability to give rise to all of the major neuronal, astrocyte and oligodendrocyte cells, and in vitro regeneration ability [37]. Due to these features, they are a promising tool for therapeutic application in ALS. Studies on animal models revealed that motor neuron degeneration activates endogenous NSC pools in the CNS to multiplicate, migrate and stimulate neurogenesis in the spinal cord as a natural defense mechanism, however the number of these cells does not seem to be adequate to fight the advancing degeneration connected with ALS [37]. In SOD1 rats, the intra-spinal application of human NSCs prolonged lifespan for more than 10 days, and had a protective impact on the number and function of the motor neurons. The visible properties assigned to the grafted NSCs
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demonstrate their integration into the spinal cord, differentiation, and formation of functional synapses with host MNs [38-39]. In a different study, the spinal lumbar injections of human NSCs into ALS rats revealed temporary amelioration on motor neuron number and function in the neighborhood of grafted cells; however, MNs located at a greater distance from the transplanted lumbar regions were not defended [40]. This result implies that, in order to gain the appropriate preclinical and clinical outcomes, cells should be injected into both the spinal and supra-spinal areas. Xu et al. (2011) supported this statement through research conducted on G93A-SOD1 rats, which demonstrated that double NSC grafts to many sites of the spinal cord extend lifespan by up to 17 days [41]. Recently, a consortium of 11 independent ALS researchers has demonstrated that the delivery of NSCs into transgenic murine models delayed the onset and progression of clinical symptoms and improved motor function. In these experiments, it was also found that 25% of the NSC-treated ALS mice had a prolonged lifespan of almost 12 months (three times longer than untreated mice) [42]. It should be emphasized that, among the functional properties of NSCs, they support the existing damaged MNs by their neurotrophic (humoral) and anti-inflammatory activities. NSCs and other stem cells produce and excrete various immunomodulatory molecules, which regulate cell migration, growth and differentiation and lead to the process of neurogenesis and angiogenesis [43-44]. One of the past reports indicated that vascular endothelial growth factor (VEGF) released from NSC-treated transgenic animals, provided a neuroprotective effect by the formation of anti-apoptotic proteins and cell lifespan-mediating molecules, as well as by the down-regulation of pro-apoptotic proteins. Furthermore, this cell transplantation support motor neuron survival and improved motor function in those affected by ALS [45-46]. Generally speaking, these preclinical studies highlighted the importance and effectiveness of NSCs in ALS therapy. The results obtained from these animal experiments have provided the
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foundation for the first clinical trials that aim to utilize the exceptional characteristics of NSCs. 3.2.Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are multipotent stromal cells that can be easily derived from various sources, such as the umbilical cord, adult bone marrow (the best described origin of MSCs), Wharton's jelly, the placenta, adipose tissue, fetal liver and others. Subsequently, MSCs give rise to osteoblasts, chondrocytes, and adipocytes [47]. MSCs have been administered as a therapeutic strategy in treating many types of diseases, including neurodegenerative disorders, and can be recognized by negative and positive profiling of different hematopoietic surface markers. Their positive impact after transplantation involves the release of neurotrophic and immunomodulatory molecules (cytokines and growth factors such as IL-6 and IL-10, TGF-β, insulin-like growth factor (IGF-1) and VEGF). When delivered regularly, they migrate to the damaged tissue sites exhibiting inflammation [48-49]. What is more, MSCs have the possibility to transdifferentiate into neuron-like cells. So far, the protocols for inducing neuron-like cell differentiation used several chemicals which are toxic and cannot be applied in humans [50]. Conversely, growth factors, such as the epidermal growth factor (EGF), VEGF and hepatocyte growth factor (HGF), are naturally secreted in the human organism and may be safety used to induce differentiation. Nevertheless, employing these neuronal cells in clinical trials, especially for the questionable formation of functional neurons, continues to raise doubts [51]. There is a need for long-term preclinical studies on in vitro differentiation and biological activity of human MSCs. Animal model studies revealed that the most promising candidates for ALS therapy are bone marrow-derived MSCs (BM-MSCs), which successfully improve the clinical and pathological features of the disease. This is associated with the availability and quantity of BM-MSCs in relation to other stem cell classes. G93A-SOD1 mice receiving intra-venous,
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intra-thecal, intra-cerebral and intra-spinal MSC grafts exhibit favorable effects on the course of ALS, namely the recovery of motor function, decreased loss of MNs and extended lifespan [31, 52-54]. In turn, the intra-spinal administration of BM-MSCs in G93A-SOD1 mice confers a range of beneficial effects on neuroinflammation, astrocytosis, and the activation of microglial cells [55-56]. MSCs, apart from the delivery of neuroprotective factors to the CNS, are also involved in the regulation of inflammation and activation of endogenous cells to participate in tissue repair. In the G93A-SOD1 mice, the intra-cerebroventricular application of BM-MSCs expressing glucagon-like peptide 1 (with antioxidant properties) prolonged lifespan for 13 days, reduced neuro-inflammation, astrocytosis, and activation of microglial cells, and delayed the onset of ALS by 15 days [57]. Moreover, the intra-muscular application of G93A-SOD1 rats with BM-MSCs expressing higher levels of glial-derived neurotrophic factor (GDNF) leads to the recovered health of MNs and lengthens lifespan by 28 days [49]. In addition, Kwon et al. (2014) showed that BM-MSCs (intrathecally delivered) exert immunomodulatory action in ALS patients via the regulation of immune cells, such as T regulatory cells, which secrete higher levels of IL-4, IL-10 and TGF-β [58]. In turn, there is evidence to suggest that human BM-MSCs intracerebroventricularly injected into ALS mice do not always bring about benefits in regard to neuroinflammatory response or motor neuron lifespan [59]. This may be related to the fact that BM-MSCs also exert their immunomodulation by inhibiting the activation and function of different cells of the specific and non-specific immune system, such as dendritic cells, T/B lymphocytes, macrophages, neutrophils and natural killer cells [60]. There are also reports concerning adipose-derived MSCs and their use in the treatment of ALS [61-62]. The systemic administration of MSC to SOD1-mutant mice at the clinical onset delayed motor deterioration by 4-6 weeks, maintained the strength and function of lumbar motor neurons, and upregulated levels of GDNF and basic FGF in the spinal cord
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[61]. From the presented preclinical data, it seems that MSCs, owing to their therapeutic plasticity, suite the complex character of ALS quite well. This feature predisposes them to being a powerful candidate for the treatment of ALS as discussed below. 3.3.Embryonic Stem Cells Embryonic Stem Cells (ESCs) are pluripotent stem cells derived from the inner cell mass of mammalian embryos at the blastocyst stage. Two characteristic features of ESCs are: the ability of unrestricted self-renewal, and the capacity of differentiating into all three germ layers, i.e. the endoderm, mesoderm, and ectoderm [63]. Using various cell-signaling molecules, ESCs can be differentiated into nerve cells such as inter-neurons [64], dopaminergic neurons [65], astrocytes [66], oligodendrocytes [67] and microglia [68]. One of the potential approaches in ALS treatment using ESCs may be the substitution of new MNs in place of degenerated ones. Previous research has demonstrated that MNs derived from ESCs may preserve molecular and functional properties after their transplantation into the spinal cords of adult rodents with motor neuron damage [69-70]. A study investigating intra-spinal transplantation of mouse ESC-derived MNs into an ALS affected SOD1 rat model carrying the G93A mutation reported temporarily improved functional effects. There were, however, no visible axonal projections to the periphery, no impact on the formation of the neuromuscular junction, no long-term influence on the lifespan of the animal and poor graft survival. Taking the above facts into account, it is doubtful that the direct replacement of lost MNs influences the course of ALS [71]. Rapid proliferation of ESCs and their easy adaptation to different environments is conducive to tumor formation after transplantation into the animal models. The tumorigenic nature of ESCs is one of the reasons preventing the clinical application of these cells with the issue remaining under investigation [72]. Furthermore, the strict regulatory requirements of human ESCs and their limited supply hold back research on the utilization of ESCs in ALS therapy.
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3.4.Induced Pluripotent Stem Cells Induced pluripotent stem cells (iPSCs) are a novel type of stem cells used for autologous grafts. They have features similar to ESCs, but with no ethical issues and reduced risk of immune rejection. However, iPSCs can be characterized by substantial variations, for instance epigenetic memories according to the source of the transformed cells. They are derived from somatic cells that had been reprogrammed to an ESC-like pluripotent state by the induction of particular genes transcribed in ESCs. iPSCs are mostly generated from human dermal fibroblasts [73] as well as from other types of cells such as blood cells and keratinocytes [74]. Previous evidence indicates that the human iPSCs can be generated to induce neural progenitor cells (iNPCs). The mentioned cells have the ability to induce astroglial differentiation and survive in the spinal cord after transplantation [75]. In addition, there are findings reporting the effective generation of MNs and NPCs from ALS patients and murine iPSCs [76-77]. Nevertheless, research is needed to determine their ability to migrate and their effective impact on the symptoms of the disease. In the spinal cord of wild-type and transgenic G93A-SOD1 rats, the transplantation of iPSC-derived neural progenitors was performed successfully and the grafted cells have survived in a large quantity. In this study, the axonal growth or synaptic formation of the delivered cells was not identified, and neither was the impact of engraftment on functional results, such as motor deficits or disease progression [78]. On the other hand, intravenous and intra-thecal administration of human iPSC-derived neural progenitors with high aldehyde dehydrogenase (ADH) activity and integrin VLA4 positivity improved neuromuscular function, the disease phenotype and survival in ALS transgenic mice [79]. Due to the early stage of iPSC reprogramming technology, there is limited information about clinical trials using these cells in treating ALS. This may be linked to the fact that iPSCs carry the risk of tumor formation. However, there is evidence that the development of maximum standardization of conditions for iPSC culturing
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and differentiation may lead to a reduced number of undifferentiated cells, which would prevent/restrict the formation of teratomas. In relation to this fact, the Takahashi group has optimized the protocol for the use of human iPSC-derived retinal pigment epithelium (RPE) cell sheets in clinical application. The above-mentioned procedure gave rise to the first clinical study aiming to transplant iPSC-derived RPE sheets into patients with age-related macular degeneration [80]. The future of iPSCs in clinical trials is more and more encouraging and carries the prospect of their use in ALS treatment and as a model in drug screening. 3.5. Human Umbilical Cord Blood Cells Human umbilical cord blood cells (hUBCs) are a heterogenous mixture of mononuclear cells that include stem cells. hUBCs characterized by low pathogenicity and immune immaturity have the capacity, under in vitro conditions, for differentiation into neural-like cells [81]. In addition, these cells, displaying the properties of progenitor/stem cells, secrete trophic and anti-inflammatory IL-10 in great quantities [81-82]. There is much evidence to show that, in animal models of ALS, intra-cerbroventricular delivery of hUSCs suppressed the progression of the disease and prolonged lifespan by secretion of anti-inflammatory cytokines and chemokines [83]. The neurotherapeutic effect was also achieved by genetically modified hUBCs (overexpressing trophic factors), which, when retro-orbitaly delivered, not only support symptomatic outcomes in ALS mice but also differentiate into astrocytes [8485]. Other studies have shown that, in the early stage of the disease, injection of hUBCs improved motor function and neuromuscular transmission, decreased the loss of MNs and extended survival of transgenic mice [86-87]. In order for the transplantation of hUBCs in a preclinical study of ALS to be effective, these cells need to be injected multiple times [82] and tracked by safe in vivo methods to reach the degenerated region of the CNS [81]. In addition to this, it has been established that iPSCs from lentiviral transductions of hUBCs
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may differentiate into functional MNs and serve as a promising tool for transplantation studies and drug modeling [88]. All the preclinical data regarding hUBC-based therapy indicates their future use in clinical studies, although scientific reports on this matter are still limited. 3.6.Olfactory Ensheathing Stem Cells Olfactory ensheathing stem cells (OESCs) are known as a specialized type of glial cells which ensheath the non-myelinated axons of olfactory neurons. In preclinical studies, OESCs were found to be a useful tool not only in the treatment of spinal cord injuries but also in ALS therapy. Research on SOD1 transgenic rats indicates that intra-spinal OEC transplantation prolongs the survival of animals through neuroprotection and remyelination [89], as well as the intracranial application of these cells improving the overall function of mutant rodents [90]. In ALS therapy, Chinese researchers performed a large number of clinical trials in which OESC transplantations brought benefits [31]. However, these studies have been criticized by other scientists because of the poorly designed clinical trial, particularly due to the large number of patients enrolled. Despite the positive effect of OESCs in ALS therapy, there are findings indicating that there were no significant objective improvements in 7 Dutch ALS patients who had undergone experimental OESC treatment in China [91]. Similarly, postmortem evaluation of brain tissue from 2 patients demonstrated transplant encasement, but with no positive effect on neuroprotection or axonal regeneration [92]. These reports suggest that there is a need to continue further preclinical research to determine the safety and efficacy of OSC therapy for ALS affected individuals. 4.
Clinical advances in stem cell therapies for ALS When considering translation of stem cell therapy from the laboratory bench to clinical
application, certain criteria have to be applied: I) an easily available origin of stem cells; II) successful transplantation of cells to the infected and damaged regions; III) the capacity of cells to survive and incorporate into host circuits. As far as the first requirement is concerned,
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the current clinical research is particularly focused on the use of NSCs and BM-MSCs. For the clinical use, stem cells should be produced according to good manufacturing practice (GMP) protocols, which aims to help eliminate obstacles encountered during the culture (e.g. microbial contamination) or expansion (e.g. genomic instability, senescence or decreased differentiation potential) of cells. It is very important that whole process of cell production is reproducible, robust and efficient. The next criterion, concerning the delivery techniques for different stem cell types in ALS therapy, includes intra-thecal, intra-spinal, intraventricular, intramuscular, and intra-arterial routes of administration. Ultimately, the last condition refers to the integrity of stem cells, which must be clinically quantifiable [93]. This results in the need to track grafted cells from the moment of their transplantation until they reach the host environment. One of the most common methods of monitoring transplanted cells is histopathological evaluation; however, such analysis is highly invasive. Nowadays, more advanced non-invasive cellular imaging techniques for visualizing engrafted cells in real-time exist. So far, optimized protocols have been prepared for stem cells, which are magnetically labeled by superparamagnetic iron oxide particles and tracked by magnetic resonance imaging (MRI). This cellular imaging technique, combined with contrast agents, has been widely used in in vitro studies, where grafted cells with efficient labeling did not exhibit impaired survival, proliferation, self-renewal or multipotency [94]. Cell integrity also depends on the progression of the disease and changes in functional status in ALS patients, which are measured using the Revised ALS Functional Rating Scale (ALSFRS-R)[93]. Additionally taken into account are also the estimated respiratory parameters and lifespan, as well as the records of a given number of assessed improvements in health as compared to control treatments.
4.1.
Clinical Application of NSCs for ALS therapy
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Due to the encouraging results of preclinical studies with NSC-based therapies, the US Food and Drug Administration approved a clinical trial using intra-spinal injection of human NSCs. In a phase I trial, 18 patients with ALS were monitored for up to 2.5 years after NSC transplantations. Twelve patients underwent unilateral or bilateral microinjections into the lumbar spinal cord, and six patients received unilateral microinjections into the cervical spinal cord. There were no observed major adverse events attributable to the surgery or cells [9596]. In November 2012, a phase II trial began, which is currently ongoing, but not recruiting new participants (ClinicalTrials.gov Identifier: NCT01730716). The aim of this study is to determine the efficiency, safety, toxicity, and maximal tolerated (safe) dose of NSCs for ALS therapy. The clinical trial, which started in July 2012 in Italy, was completed in December 2015 (ClinicalTrials.gov Identifier: NCT01640067). The phase I trial was performed on six ALS patients receiving unilateral or bilateral NSC microinjections into the lumbar spinal cord. The initial results demonstrated that, during the 18-month follow-up period after cell transplantations, surgical procedures did not cause an increase of disease progression. Moreover, two patients showed a transitory improvement in the ambulation sub-score on the ALS-FRS-R scale, another patient showed improvement in the Medical Research Council score for tibialis anterior muscles. Mazzini et al. (2015) described a safe cell strategy, which will allow for the treatment of larger patient populations in later-phases of ALS clinical studies. At the moment, the same research group in Italy is carrying out research on 12 ALS patients who underwent intra-spinal NSC injections into the cervical spinal cord [97]. 4.2.Clinical Application of BM-MSCs for ALS therapy Many cell-based clinical studies for ALS are underway using BM-MSCs. Most of them are in phase I/II, aimed to demonstrate the safety and feasibility of transplantation. In Israel, a phase I/II clinical trial was conducted on 15 patients with MS and 19 patients with ALS, confirming the safety and immunological effect of intrathecal and
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intravenous delivery of MSCs. The 19 months of follow-up did not show any side effects in the course of the disease and indicated prolonged stabilization, or even improvements in certain patients, connected with a beneficial immunomodulatory effect [98]. A Korean research group has demonstrated that two repeated intrathecal autologous BM-MSCs injections (phase I trial) were safe and feasible for at least 12 months in 7 patients. No adverse effects were observed [99]. Apart from research concerning the safety of use of autologous MSC transplantation in a clinical trial in Turkey, an assessment of the secondary outcome in a study on 13 patients receiving MSC transplants into the cervical spinal cord showed promising effects. Nine of these 13 patients demonstrated motor improvement after cell delivery [100]. A research team in Spain evaluated 11 patients following intra-spinal MSC grafts. No serious adverse events were noted, and the number of MNs was greater in the areas of grafting [101]. In the above-mentioned cases, an alternative approach has also been used in which, instead of injecting MSCs, endogenous MSCs were mobilized in ALS patients by a granulocyte colony-stimulating factor. The trials carried out in Canada and Italy have shown the therapy to be safe, revealed mobilization of MSCs and confirmed anti-inflammatory responses in the spinal cord [102-103]. Another phase I clinical trial was carried out in Italy on 10 patients suffering from ALS. Autologous BM-MSCs were isolated, expanded in vitro, and suspended in autologous CSF. Subsequently, cells were delivered into the spinal cord at high thoracic level. The patients were subjected to observation, both before and after transplantation, by clinical, psychological, neuroradiological and neurophysiological evaluations. The study also indicated MSC transplantation to be safe and well-tolerated by ALS patients [104-107]. However, all ongoing clinical trials using BM-MSCs for the treatment of ALS patients are in phase I and/or II (Table 1). Therefore, more research is required in this direction.
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5. Conclusions The limited number of clinical research indicates that NSCs and MSCs may bring be most effective positive effects in the treatment of ALS (Table 1). However, there are no clear guidelines for the optimal number of grafted cells, route of delivery, or the enrollment of patients for trials. It is also essential to have a certainty of the graft lifespan in order to reach permanent efficacy. The results of preclinical research indicate that transplanted human cells in animal models can be identified by immunohistochemistry using specific markers for human cells. A challenge for stem cell therapeutic approaches is also a need for the reliable tracking of grafted cells in clinical trials. Currently, although it is highly invasive and requires various tissue biopsies, histopathological evaluation is the “basic” method of monitoring transplanted cells. In recent years, however, advanced non-invasive cellular imaging techniques for visualizing engrafted cells in real time are being used more and more frequently. One of these advanced methods is MRI, which involves labeling cells with super paramagnetic iron oxide nanoparticles or with reporter genes and allows for an understanding of how the implanted cells migrate into or within the spinal cord, and their effect on degenerated motor neurons. Lately, a large portion of research is focused on stem cell-derived EVs, which mediate both immunomodulatory and neuroprotective effects, and might be engaged in the regeneration of damaged motor neurons. However, more studies on EVs need to be done to overcame problems connected with safety, manufacturing and availability, which would enable their use in clinical studies. It is therefore necessary to continue research and strive towards an effective cell-based therapy for the treatment of ALS.
19
Acknowledgements This is supported by the National Centre for Research and Development Grant STRATEGMED1/234261/2NCBR/2014.
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Table 1. List of past and present clinical stem cell trials in ALS
Ordinal number
Type of stem cells
Specificity of cells
Application method
Status of trial
Trial institution/ sponsor and country
NCT number and duration period
1.
Neuronal Stem Cells (NSCs)
Human fetal spinal cord
Phase II, not verified status
2.
Mesenchymal Stem Cells (MSCs) Hematopoietic Stem Cells (HSCs)
Umbilical cord
Intraspinal Intrathecal Intrathecal
Neuralstem Inc., United States General Hospital of Chinese Armed Police Forces, China Hospital Universitario Dr. Jose E. Gonzalez, Mexico
NCT01730716 2013-2016 NCT01494480 2012-2015 NCT01933321 2012-2014
3.
4.
MSCs
Autologous, derived from patient bone marrow and mobilized to blood Autologous, derived from
Intrathecal
Phase II, patient’s recruitment Phase II/III, complete
Intrathecal
Phase I, patient’s recruitment
University of Warmia and Mazury, Poland
NCT02881489 2015-2018
Phase I, ongoing, but without patient’s recruitment Phase I/II, study withdrawn Phase I/II, not verified status Phase I, complete
Neuralstem Inc., United States
NCT01348451 2009-2016
patient bone marrow
5.
NSCs
Human fetal spinal cord
Spinal cord injections
6.
MSCs
Autologous
Intrathecal
7.
Mononuclear cells (MNCs)
8.
Stem Cells (SCs)
9.
MSCs
Autologous, derived from patient bone marrow Autologous, derived from patient adipose tissue Autologous, derived from patient adipose tissue
Intraspinal Intrathecal Intravenous Intracerebral Intrathecal
10.
MSCs
Autologous
Intrathecal
11.
MSCs
Allogeneic, derived from Wharton’s jelly umbilical cord
Intrathecal
Phase I, ongoing, but without patient’s recruitment Phase I, patient’s recruitment Phase I, patient’s recruitment
Alzahra Hospital, Iran
NCT02116634 2015-2017 Hospital Universitario NCT01254539 Virgen de la Arrixaca, Spain 2010-2015 China Medical University NCT02383654 Hospital, China 2015-2016 Anthony J. Windebank, NCT01609283 Mayo Clinic, United States 2012-2017 Hospital e Maternidade Dr. Christóvão da Gama, Brazil University of Warmia and Mazury, Poland
NCT02987413 2014-2017 NCT02881476 2015-2018
33
12.
Stem/progenitor cells
13.
MNCs
14.
Phase I, patient’s recruitment Phase I/II, complete
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow
Intrathecal
NSCs
Human fetal
Phase I, complete
15.
MNCs
16.
SCs
17.
MSCs
Intraventricular
18.
MSCs
Intrathecal
Phase I, patient’s recruitment Phase I, complete
19.
MNCs
Intrathecal
Phase I, complete
20.
SCs
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Adipose tissue is derived from healthy donors. Autologous, derived from patient adipose tissue Autologous, derived from patient bone marrow N/A
Spinal cord injections Intrathecal intramuscular Intrathecal
N/A
N/A, patient’s recruitment
21.
MNCs
Intramuscular
22.
Phase I, patient’s recruitment Phase I, study not open
23.
Neuronal Progenitor Cells releasing Glial cell-derived neurotrophic factor SCs
Autologous, derived from patient bone marrow Human fetal cortex
Intrathecal
Phase I/II, complete
24.
SCs
Intrathecal
Phase I, not verified status
25.
Glial Restricted Progenitor Cells (GRPs) MSCs
Autologous, derived from patient bone marrow Allogenic, derved from donor bone marrow N/A
Spinal cord injections Intraventricular
Phase I/II, study not open
26.
Autologous, derived from patient bone marrow
Intraspinal
Spinal cord injections
Phase II, complete Phase 1, study suspended
Phase I, complete
Pomeranian Medical University Szczecin, Poland Fundacion para la Formacion e Investigacion Sanitarias de la Region de Murcia, Spain Azienda Ospedaliera Santa Maria, Italy Neurogen Brain and Spine Institute, India TCA Cellular Therapy, United States Royan Institute, Iran Mayo Clinic, United States Neurogen Brain and Spine Institute, India University of Michigan, United States Red de Terapia Celula, Spain Cedars-Sinai Medical Center, United States
NCT02193893 2010-2017 NCT00855400 2007-2010
NCT01640067 2011-2015 NCT01984814 2008-2013 NCT01082653 2010-2014 NCT02492516 2014-2016 NCT01142856 2010-2011 NCT02242071 2008-2016 NCT02058732 2013-2019 NCT02286011 2014-2018 NCT02943850 2017-2019
Corestem, Inc., Korea
NCT01363401 2011-2013 Hanyang University Seoul NCT01758510 Hospital, Korea 2012-2017 Q Therapeutics, Inc., United NCT02478450 States 2017-2020 Royan Institute, Iran NCT01759797 2013-2014
34
Intraventricular
Phase I/II, patient’s recruitment Phase I, study withdrawn
University of Sao Paulo General Hospital, Brazil Royan Institute, Iran
Intrathecal
Phase I, complete
Royan Institute, Iran
Intravenous injections
Phase I/II, patient’s recruitment
Human Somatic Cells
N/A
N/A
Biopsy from patient skin
N/A
N/A
MSCs releasing Neurotrophic Factors (NTFs)
Autologous, derived from patient adipose tissue
Intramuscular Intrathecal
Phase II, complete
34.
MSCs releasing NTFs
intramuscular
Phase II, not verified status
35.
MSCs releasing NTFs
Autologous, derived from patient adipose tissue Autologous, derived from patient adipose tissue
Intramuscular intrathecal
Phase I/II, complete
Andalusian Initiative for Advanced Therapies Fundación Pública Andaluza Progreso y Salud, Spain Hadassah Medical NCT00801333 Organization, Israel 2008-2020 Institut Pasteur, France NCT01639391 2012-2014 Brainstorm NCT02017912 Cell Therapeutics, United 2014-2016 States Hadassah Medical NCT01777646 Organization, Israel 2012-2014 Hadassah Medical NCT01051882 Organization, Israel 2011-2013
27.
MSCs
28.
MSCs
29.
MSCs
30.
MSCs
31.
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Autologous, derived from patient adipose tissue
Inreathecal
Induced Pluripotent Stem Cells (iPSCs) IPSCs, Fibroblasts
33.
32.
NCT02917681 2016-2019 NCT01759784 2014-2017 NCT01771640 2013-2014 NCT02290886 2014-2021
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S0736-5748(16)30037-5 http://dx.doi.org/doi:10.1016/j.ijdevneu.2017.01.003 DN 2151
To appear in:
Int. J. Devl Neuroscience
Received date: Revised date: Accepted date:
23-2-2016 6-1-2017 6-1-2017
Please cite this article as: Czarzasta, Joanna, Habich, Aleksandra, Siwek, Tomasz, Czapli´nski, Adam, Maksymowicz, Wojciech, Wojtkiewicz, Joanna, Stem cells for ALS: an overview of possible therapeutic approaches.International Journal of Developmental Neuroscience http://dx.doi.org/10.1016/j.ijdevneu.2017.01.003
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Stem cells for ALS: an overview of possible therapeutic approaches Joanna Czarzasta1*, Aleksandra Habich2, Tomasz Siwek2, Adam Czapliński2,3, Wojciech Maksymowicz2, Joanna Wojtkiewicz1, 4, 5
1
Department of Pathophysiology, 2Department of Neurology and Neurosurgery, Faculty
of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland 3
Neurocentrum Bellevue, Neurology, Zurich, Switzerland
4
Laboratory of Regenerative Medicine, University of Warmia and Mazury, Olsztyn, Poland
5
Foundation for nerve cells regeneration, Olsztyn, Poland
* Corresponding author: Tel: 0048(89)5246133; fax: 0048(89)5277004 E-mail address: [email protected] (J. Czarzasta)
Department of Pathophysiology, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland
Highlights:
1, Amyotrophic lateral sclerosis (ALS) is an unusual, fatal, neurodegenerative disorder
leading to the loss of motor neurons.
2. Multiple factors are contributed to the progression of ALS.
3. Stem cells are the most promising tool used in the preclinical and clinical trials for ALS. In this review we would like to discuss therapeutic properties of this cells and introduce completed and ongoing clinical trials about stem cell therapy for ALS.
1
Abstract Amyotrophic lateral sclerosis (ALS) is an unusual, fatal, neurodegenerative disorder leading to the loss of motor neurons. After diagnosis, the average lifespan ranges from 3 to 5 years, and death usually results from respiratory failure. Although the pathogenesis of ALS remains unclear, multiple factors are thought to contribute to the progression of ALS, such as network interactions between genes, environmental exposure, impaired molecular pathways and many others. The neuroprotective properties of neural stem cells (NSCs) and the paracrine signaling of mesenchymal stem cells (MSCs) have been examined in multiple pre-clinical trials of ALS with promising results. The data from these initial trials indicate a reduction in the rate of disease progression. The mechanism through which stem cells achieve this reduction is of major interest. Here, we review the to-date pre-clinical and clinical therapeutic approaches employing stem cells, and discuss the most promising ones.
Key words: amyotrophic lateral sclerosis, risk factors, stem cells therapy,
1. Introduction Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a progressive neurodegenerative disorder affecting upper and lower motor neurons (MNs) in the cerebral cortex, brainstem and spinal cord [1]. Hence, the signs of damage to MNs are both at the peripheral (e.g. atrophy) and central (e.g. spasticity) level. To date, there has been no effective treatment for ALS; however, there is one US Food and Drug Administration approved treatment - Riluzole, which has a protective effect on motor neuron degeneration
2
through excitotoxicity by blocking the release of glutamate, with has an impact on NMDA or AMPA receptors. This neuroprotective agent specifically inhibits sodium channels and modulates the GABAergic system, which indicates that it affects many pathogenic mechanisms [2]. The drug may extend patient survival by a few months, though only if used in the early stages of the disease [3]. The majority of patients die within 3 to 5 years after diagnosis, usually due to respiratory failure, while some may survive even up to ten years. Prevalence ranges from 1-2 to 4-6 per 100,000 individuals per year, and thus ALS is classified as a rare disease. New theories consider it to be a multi-systemic illness, rather than a disorder involving only motor neurons. Approximately 10% of patients suffering from ALS (motor symptoms predominate) exhibited symptoms of frontotemporal dementia (FTD) [4]. ALS is a very individual disorder with many factors leading to its onset. Some of them, so-called “predictive factors”, concern mainly age, sex and family history. The average age of incidence for ALS is 55-60 years, with the disorder affecting men more often than women [5]. Most cases of ALS are sporadic (sALS) with no family history, while only 10-15% of cases are familial (fALS), with dominant inheritance [6]. The etiopathogenesis and mechanisms that underlie ALS are not fully understood, but many risk factors have been considered, including genetic, environmental and epigenetic agents, as well as substantial changes in the motor neuron microenvironment [7]. Due to the modest effect of the only available drug in the treatment of ALS, the development of new and effective strategies has high priority and a variety of approaches are in various stages of development and clinical trials. Possible treatments are associated with anti-glutamatergic, anti-oxidant, mitochondrial, and antiinflammatory agents [8]. In order to deliver support to all of these trophic factors, gene therapy is also carried out. In recent years, stem cell therapy in ALS treatment has gained a lot of attention because of the multifaceted background of this disorder. The goal of this review is
3
to: (1) introduce the existing risk factors involved in amyotrophic lateral sclerosis, and (2) discuss potential therapeutic stem cell strategies. 2. Suggested mechanisms responsible for ALS onset In the last two decades, several possible mechanisms and agents related to the origin, onset and development of ALS have been investigated. Despite the fact that the majority of ALS forms are sporadic, a wide range of genetic mutations have been identified in the autosomal dominant familial type of this disease [9]. In 20% of fALS patients, the most frequent mutation is linked to the gene encoding Cu/Zn superoxide dismutase 1 (SOD1) [5]. This mutation is connected with an antioxidant defense system against reactive oxygen species (ROS), which consist of three isoforms of SOD, including cytoplasmic Cu/ZnSOD (SOD1). The antioxidant defense system plays a critical role in inhibiting oxidative inactivation of nitric oxide. Indisputable evidence shows that oxidative stress is one of the mechanisms leading to the MN degeneration by an increase in markers of oxidative damage in the spinal cord and cerebrospinal fluid (CSF) of ALS patients [10]. Although oxidative damage in the pathogenesis of ALS is extensive, the precise mechanism(s) that results in elevated levels of ROS have not been fully explained. Other common genetic abnormalities causing both sALS and fALS include those involving the 43-kDa TAR DNA-binding protein (TARDP) gene, chromosome 9 open reading frame 72 (C9ORF72) gene and several others [5]. However, so far no ALS gene has been exclusively linked to only the ALS motor phenotype, indicating that ALS is a multi-system neurodegenerative disease with a propensity to affect the motor system. Apart from previous findings concerning the existence of the genetic predisposition to ALS, there are also environmental, so-called “non-lifestyle factors”, and lifestyle factors associated with the pathogenesis of this disorder. Among the factors not connected to lifestyle are neurotropic viruses and cyanobacterial toxins, magnetic fields, the metalloid selenium, and heavy metals as well as certain chemicals [11]. One of the examined
4
heavy metals in ALS patients is lead, which was found to be in higher levels in several different tissues of people suffering from ALS [12-15]. It was also shown that work and home exposure to some chemicals, such as pesticides and fertilizers, appears to be greater in ALS cases as compared to control groups matched for age and gender [16]. In addition, the increased risk of ALS is associated with lifestyle factors, such as smoking, diet, history of trauma and intense physical activity [17]. The relationship between the genetic predisposition and environmental influence indicates the involvement of epigenetic factors in the pathogenesis of ALS. Epigenetic changes refer mainly to covalent post-transcriptional modifications of DNA, histone and chromatin remodeling, RNA editing, and non-coding RNAs. In consequence, the abovementioned alterations may regulate the gene expression and function [18]. All of these mechanisms manage neuronal development, plasticity, as well as aging, and are very important in maintaining cellular homeostasis [19]. ALS is not only related to genetic, environmental and epigenetic factors, but also to the changes in the microenvironment of motor neurons. Multiple events may contribute to the pathogenesis of ALS, such as excitotoxicity, oxidative stress, mitochondrial dysfunction, protein aggregation, dysregulation of RNA processing, neuro-inflammation, disruption of axonal transport and glial cell dysfunction [5, 20-21]. Of the above-mentioned events, neuroinflammation is a major player in the course of ALS, and is characterized by astrocyte and microglial activation, T cell infiltration, and the overproduction of proinflammatory cytokines and other factors. Astrocyte activation during the neuroinflammatory process leads to the secretion of cytokines and growth factors. It is known that astrocyte-derived transforming growth factor/TGF/-β1 fulfills an anti-inflammatory role in the neuroprotective inflammatory reaction in transgenic mice by regulating microglial activation, T-cell numbers, and interferon /IFN/-γ and interleukin /IL/-4 balance [22]. What is more, microglial cells, as
5
primary mediators of neuroinflammation, may be transformed into phagocytes upon damage to the cell in the resting phase, or release inflammatory molecules in the active phase. Microglia may be activated in the early stages of ALS, or in themselves triggers the disease and plays a dual function, either protective or harmful. T lymphocytes, on the other hand, by involvement in the inflammation, have a contrarily protective function at early stages of ALS, though this effect is not sustained. They are also linked with microglial functions by inhibiting the harmful impact of microglia or enhancing the neuroprotective influence of these cells. Among the many components/molecules engaged in the inflammatory response, cytokines such as IL-1β, IL-7, IL-9, IL-12, IL-17, tumor necrosis factor-α and IFN-γ, as well as also chemokines were upregulated in CSF of ALS patients [23]. Similarly to microglia, cytokines/chemokines may exert a harmful or protective effect. Correlating their level with clinical ALS status, some of them may indicate worsened neurological function, while others may slow down the progression of the disease by improving the functional performance of the patient. As it had been mentioned above, ALS is a multi-factorial and multi-systemic disease, the effects of which include a reduction of blood-brain and blood-spinal cord barrier (BBB/BSCB) integrity. Under physiological conditions, BBB and BSCB are responsible for the control of brain homeostasis, regulation of influx and efflux transport, and protection from damage by their own specialized multicellular structure, such as the capillary endothelium, cell-tight endothelial junctions, capillary basement membranes and astrocytes [24]. Studies carried out on SOD1 mice and ALS patients showed that BBB and BSCB integrity is disrupted by damage to endothelial cell capillaries, astrocyte end-feet, perivascular edema, alterations to basement membrane components, etc. [24-25]. It seems that the role of decreased BBB and BSCB integrity in ALS pathogenesis still needs to be explained; however, there is evidence to suggest that, in the mice model, BSCB disruption occurs at an early stage of the disease and that restoring barrier integrity delays the disease process [26].
6
Additionally, not only neuroinflammation but also the immune response may play a crucial role in the pathological progression of ALS [27]. During the onset of symptoms and course of the disease, alterations in the structure, quantity and role of immune cells have been found outside and within the central nervous system (CNS) [28]. It has been shown that peripheral blood lymphocytes of ALS patients have a different functionality, presumably because of differences in the immune response depending on the stage of the disease. Extensive lymphocyte proliferation in ALS patients with mitogen induction may be the result of autoimmunity alterations, while non-inducible proliferation in such patients may point to an immune deficiency [29]. During ALS progression, changes occur not only in lymphocyte proliferation, but also in the systemic pro-inflammatory state and in the antioxidant system. Higher concentrations of pro-inflammatory IL-6, IL-8 and nitrite, as well as reduced antioxidant glutathione levels, might suggest adaptive immune responses dependent on the stage of disease in the effected individuals [30]. 3. Preclinical advances in stem cell therapies for ALS In spite of the many years of trials and tests seeking an effective drug or therapeutic strategy, a method of controlling or inhibiting ALS has not yet been established. As it was previously mentioned, Riluzole is the only approved drug, prolonging patient survival for only a very short period. The future of preclinical and clinical trials is particularly linked to genetic engineering and stem cell therapy. In this review, we focus solely on the available stem cell types and their application in ALS treatment. The main distinguishing features of individual cell populations are their source of origin, accessibility, and potential for differentiation [31]. Due to their unique features, stem cells are an extremely attractive tool in ALS therapy as they may offer neuro-protection by exerting a paracrine effect and replacing degenerated neural cells [32]. 3.1.Neural Stem Cells
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Neural Stem Cells (NSCs) represent a population of progenitor cells located in the developing and mature CNS specifically in neurogenic areas of the brain, such as the subventricular and sub-granular zone [33-34]. The neurogenic niche provides a special microenvironment that determines the lifespan and fate of NSCs. This is related to the paracrine effect of these cells, including cell–cell contact and interaction with specific components of the extracellular matrix. Also, these cells in an undifferentiated form may exert “bystander” action in regard to cell replacement in inflammatory CNS disorders. Such an effect occurs when NSCs provide neurotrophic support in response to stimuli elicited by T cells within CNS, at the level of atypical perivascular areas of the multiple sclerosis (MS) animal model [35]. It needs to be highlighted that NSCs secrete extracellular vesicles (EVs) which communicate with the host’s immune system and mediate a neuroprotective and immunomodulatory effect via release their components (lipids, proteins and nucleic acids) [36]. If EVs pass the BBB and get into nerve cells during the inflammatory process, they could heal these cells; however, such cellular and molecular mechanisms have not yet been fully elucidated. NSCs are characterized by the high potential of self-renewal, properties of multipotency, i.e., the ability to give rise to all of the major neuronal, astrocyte and oligodendrocyte cells, and in vitro regeneration ability [37]. Due to these features, they are a promising tool for therapeutic application in ALS. Studies on animal models revealed that motor neuron degeneration activates endogenous NSC pools in the CNS to multiplicate, migrate and stimulate neurogenesis in the spinal cord as a natural defense mechanism, however the number of these cells does not seem to be adequate to fight the advancing degeneration connected with ALS [37]. In SOD1 rats, the intra-spinal application of human NSCs prolonged lifespan for more than 10 days, and had a protective impact on the number and function of the motor neurons. The visible properties assigned to the grafted NSCs
8
demonstrate their integration into the spinal cord, differentiation, and formation of functional synapses with host MNs [38-39]. In a different study, the spinal lumbar injections of human NSCs into ALS rats revealed temporary amelioration on motor neuron number and function in the neighborhood of grafted cells; however, MNs located at a greater distance from the transplanted lumbar regions were not defended [40]. This result implies that, in order to gain the appropriate preclinical and clinical outcomes, cells should be injected into both the spinal and supra-spinal areas. Xu et al. (2011) supported this statement through research conducted on G93A-SOD1 rats, which demonstrated that double NSC grafts to many sites of the spinal cord extend lifespan by up to 17 days [41]. Recently, a consortium of 11 independent ALS researchers has demonstrated that the delivery of NSCs into transgenic murine models delayed the onset and progression of clinical symptoms and improved motor function. In these experiments, it was also found that 25% of the NSC-treated ALS mice had a prolonged lifespan of almost 12 months (three times longer than untreated mice) [42]. It should be emphasized that, among the functional properties of NSCs, they support the existing damaged MNs by their neurotrophic (humoral) and anti-inflammatory activities. NSCs and other stem cells produce and excrete various immunomodulatory molecules, which regulate cell migration, growth and differentiation and lead to the process of neurogenesis and angiogenesis [43-44]. One of the past reports indicated that vascular endothelial growth factor (VEGF) released from NSC-treated transgenic animals, provided a neuroprotective effect by the formation of anti-apoptotic proteins and cell lifespan-mediating molecules, as well as by the down-regulation of pro-apoptotic proteins. Furthermore, this cell transplantation support motor neuron survival and improved motor function in those affected by ALS [45-46]. Generally speaking, these preclinical studies highlighted the importance and effectiveness of NSCs in ALS therapy. The results obtained from these animal experiments have provided the
9
foundation for the first clinical trials that aim to utilize the exceptional characteristics of NSCs. 3.2.Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are multipotent stromal cells that can be easily derived from various sources, such as the umbilical cord, adult bone marrow (the best described origin of MSCs), Wharton's jelly, the placenta, adipose tissue, fetal liver and others. Subsequently, MSCs give rise to osteoblasts, chondrocytes, and adipocytes [47]. MSCs have been administered as a therapeutic strategy in treating many types of diseases, including neurodegenerative disorders, and can be recognized by negative and positive profiling of different hematopoietic surface markers. Their positive impact after transplantation involves the release of neurotrophic and immunomodulatory molecules (cytokines and growth factors such as IL-6 and IL-10, TGF-β, insulin-like growth factor (IGF-1) and VEGF). When delivered regularly, they migrate to the damaged tissue sites exhibiting inflammation [48-49]. What is more, MSCs have the possibility to transdifferentiate into neuron-like cells. So far, the protocols for inducing neuron-like cell differentiation used several chemicals which are toxic and cannot be applied in humans [50]. Conversely, growth factors, such as the epidermal growth factor (EGF), VEGF and hepatocyte growth factor (HGF), are naturally secreted in the human organism and may be safety used to induce differentiation. Nevertheless, employing these neuronal cells in clinical trials, especially for the questionable formation of functional neurons, continues to raise doubts [51]. There is a need for long-term preclinical studies on in vitro differentiation and biological activity of human MSCs. Animal model studies revealed that the most promising candidates for ALS therapy are bone marrow-derived MSCs (BM-MSCs), which successfully improve the clinical and pathological features of the disease. This is associated with the availability and quantity of BM-MSCs in relation to other stem cell classes. G93A-SOD1 mice receiving intra-venous,
10
intra-thecal, intra-cerebral and intra-spinal MSC grafts exhibit favorable effects on the course of ALS, namely the recovery of motor function, decreased loss of MNs and extended lifespan [31, 52-54]. In turn, the intra-spinal administration of BM-MSCs in G93A-SOD1 mice confers a range of beneficial effects on neuroinflammation, astrocytosis, and the activation of microglial cells [55-56]. MSCs, apart from the delivery of neuroprotective factors to the CNS, are also involved in the regulation of inflammation and activation of endogenous cells to participate in tissue repair. In the G93A-SOD1 mice, the intra-cerebroventricular application of BM-MSCs expressing glucagon-like peptide 1 (with antioxidant properties) prolonged lifespan for 13 days, reduced neuro-inflammation, astrocytosis, and activation of microglial cells, and delayed the onset of ALS by 15 days [57]. Moreover, the intra-muscular application of G93A-SOD1 rats with BM-MSCs expressing higher levels of glial-derived neurotrophic factor (GDNF) leads to the recovered health of MNs and lengthens lifespan by 28 days [49]. In addition, Kwon et al. (2014) showed that BM-MSCs (intrathecally delivered) exert immunomodulatory action in ALS patients via the regulation of immune cells, such as T regulatory cells, which secrete higher levels of IL-4, IL-10 and TGF-β [58]. In turn, there is evidence to suggest that human BM-MSCs intracerebroventricularly injected into ALS mice do not always bring about benefits in regard to neuroinflammatory response or motor neuron lifespan [59]. This may be related to the fact that BM-MSCs also exert their immunomodulation by inhibiting the activation and function of different cells of the specific and non-specific immune system, such as dendritic cells, T/B lymphocytes, macrophages, neutrophils and natural killer cells [60]. There are also reports concerning adipose-derived MSCs and their use in the treatment of ALS [61-62]. The systemic administration of MSC to SOD1-mutant mice at the clinical onset delayed motor deterioration by 4-6 weeks, maintained the strength and function of lumbar motor neurons, and upregulated levels of GDNF and basic FGF in the spinal cord
11
[61]. From the presented preclinical data, it seems that MSCs, owing to their therapeutic plasticity, suite the complex character of ALS quite well. This feature predisposes them to being a powerful candidate for the treatment of ALS as discussed below. 3.3.Embryonic Stem Cells Embryonic Stem Cells (ESCs) are pluripotent stem cells derived from the inner cell mass of mammalian embryos at the blastocyst stage. Two characteristic features of ESCs are: the ability of unrestricted self-renewal, and the capacity of differentiating into all three germ layers, i.e. the endoderm, mesoderm, and ectoderm [63]. Using various cell-signaling molecules, ESCs can be differentiated into nerve cells such as inter-neurons [64], dopaminergic neurons [65], astrocytes [66], oligodendrocytes [67] and microglia [68]. One of the potential approaches in ALS treatment using ESCs may be the substitution of new MNs in place of degenerated ones. Previous research has demonstrated that MNs derived from ESCs may preserve molecular and functional properties after their transplantation into the spinal cords of adult rodents with motor neuron damage [69-70]. A study investigating intra-spinal transplantation of mouse ESC-derived MNs into an ALS affected SOD1 rat model carrying the G93A mutation reported temporarily improved functional effects. There were, however, no visible axonal projections to the periphery, no impact on the formation of the neuromuscular junction, no long-term influence on the lifespan of the animal and poor graft survival. Taking the above facts into account, it is doubtful that the direct replacement of lost MNs influences the course of ALS [71]. Rapid proliferation of ESCs and their easy adaptation to different environments is conducive to tumor formation after transplantation into the animal models. The tumorigenic nature of ESCs is one of the reasons preventing the clinical application of these cells with the issue remaining under investigation [72]. Furthermore, the strict regulatory requirements of human ESCs and their limited supply hold back research on the utilization of ESCs in ALS therapy.
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3.4.Induced Pluripotent Stem Cells Induced pluripotent stem cells (iPSCs) are a novel type of stem cells used for autologous grafts. They have features similar to ESCs, but with no ethical issues and reduced risk of immune rejection. However, iPSCs can be characterized by substantial variations, for instance epigenetic memories according to the source of the transformed cells. They are derived from somatic cells that had been reprogrammed to an ESC-like pluripotent state by the induction of particular genes transcribed in ESCs. iPSCs are mostly generated from human dermal fibroblasts [73] as well as from other types of cells such as blood cells and keratinocytes [74]. Previous evidence indicates that the human iPSCs can be generated to induce neural progenitor cells (iNPCs). The mentioned cells have the ability to induce astroglial differentiation and survive in the spinal cord after transplantation [75]. In addition, there are findings reporting the effective generation of MNs and NPCs from ALS patients and murine iPSCs [76-77]. Nevertheless, research is needed to determine their ability to migrate and their effective impact on the symptoms of the disease. In the spinal cord of wild-type and transgenic G93A-SOD1 rats, the transplantation of iPSC-derived neural progenitors was performed successfully and the grafted cells have survived in a large quantity. In this study, the axonal growth or synaptic formation of the delivered cells was not identified, and neither was the impact of engraftment on functional results, such as motor deficits or disease progression [78]. On the other hand, intravenous and intra-thecal administration of human iPSC-derived neural progenitors with high aldehyde dehydrogenase (ADH) activity and integrin VLA4 positivity improved neuromuscular function, the disease phenotype and survival in ALS transgenic mice [79]. Due to the early stage of iPSC reprogramming technology, there is limited information about clinical trials using these cells in treating ALS. This may be linked to the fact that iPSCs carry the risk of tumor formation. However, there is evidence that the development of maximum standardization of conditions for iPSC culturing
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and differentiation may lead to a reduced number of undifferentiated cells, which would prevent/restrict the formation of teratomas. In relation to this fact, the Takahashi group has optimized the protocol for the use of human iPSC-derived retinal pigment epithelium (RPE) cell sheets in clinical application. The above-mentioned procedure gave rise to the first clinical study aiming to transplant iPSC-derived RPE sheets into patients with age-related macular degeneration [80]. The future of iPSCs in clinical trials is more and more encouraging and carries the prospect of their use in ALS treatment and as a model in drug screening. 3.5. Human Umbilical Cord Blood Cells Human umbilical cord blood cells (hUBCs) are a heterogenous mixture of mononuclear cells that include stem cells. hUBCs characterized by low pathogenicity and immune immaturity have the capacity, under in vitro conditions, for differentiation into neural-like cells [81]. In addition, these cells, displaying the properties of progenitor/stem cells, secrete trophic and anti-inflammatory IL-10 in great quantities [81-82]. There is much evidence to show that, in animal models of ALS, intra-cerbroventricular delivery of hUSCs suppressed the progression of the disease and prolonged lifespan by secretion of anti-inflammatory cytokines and chemokines [83]. The neurotherapeutic effect was also achieved by genetically modified hUBCs (overexpressing trophic factors), which, when retro-orbitaly delivered, not only support symptomatic outcomes in ALS mice but also differentiate into astrocytes [8485]. Other studies have shown that, in the early stage of the disease, injection of hUBCs improved motor function and neuromuscular transmission, decreased the loss of MNs and extended survival of transgenic mice [86-87]. In order for the transplantation of hUBCs in a preclinical study of ALS to be effective, these cells need to be injected multiple times [82] and tracked by safe in vivo methods to reach the degenerated region of the CNS [81]. In addition to this, it has been established that iPSCs from lentiviral transductions of hUBCs
14
may differentiate into functional MNs and serve as a promising tool for transplantation studies and drug modeling [88]. All the preclinical data regarding hUBC-based therapy indicates their future use in clinical studies, although scientific reports on this matter are still limited. 3.6.Olfactory Ensheathing Stem Cells Olfactory ensheathing stem cells (OESCs) are known as a specialized type of glial cells which ensheath the non-myelinated axons of olfactory neurons. In preclinical studies, OESCs were found to be a useful tool not only in the treatment of spinal cord injuries but also in ALS therapy. Research on SOD1 transgenic rats indicates that intra-spinal OEC transplantation prolongs the survival of animals through neuroprotection and remyelination [89], as well as the intracranial application of these cells improving the overall function of mutant rodents [90]. In ALS therapy, Chinese researchers performed a large number of clinical trials in which OESC transplantations brought benefits [31]. However, these studies have been criticized by other scientists because of the poorly designed clinical trial, particularly due to the large number of patients enrolled. Despite the positive effect of OESCs in ALS therapy, there are findings indicating that there were no significant objective improvements in 7 Dutch ALS patients who had undergone experimental OESC treatment in China [91]. Similarly, postmortem evaluation of brain tissue from 2 patients demonstrated transplant encasement, but with no positive effect on neuroprotection or axonal regeneration [92]. These reports suggest that there is a need to continue further preclinical research to determine the safety and efficacy of OSC therapy for ALS affected individuals. 4.
Clinical advances in stem cell therapies for ALS When considering translation of stem cell therapy from the laboratory bench to clinical
application, certain criteria have to be applied: I) an easily available origin of stem cells; II) successful transplantation of cells to the infected and damaged regions; III) the capacity of cells to survive and incorporate into host circuits. As far as the first requirement is concerned,
15
the current clinical research is particularly focused on the use of NSCs and BM-MSCs. For the clinical use, stem cells should be produced according to good manufacturing practice (GMP) protocols, which aims to help eliminate obstacles encountered during the culture (e.g. microbial contamination) or expansion (e.g. genomic instability, senescence or decreased differentiation potential) of cells. It is very important that whole process of cell production is reproducible, robust and efficient. The next criterion, concerning the delivery techniques for different stem cell types in ALS therapy, includes intra-thecal, intra-spinal, intraventricular, intramuscular, and intra-arterial routes of administration. Ultimately, the last condition refers to the integrity of stem cells, which must be clinically quantifiable [93]. This results in the need to track grafted cells from the moment of their transplantation until they reach the host environment. One of the most common methods of monitoring transplanted cells is histopathological evaluation; however, such analysis is highly invasive. Nowadays, more advanced non-invasive cellular imaging techniques for visualizing engrafted cells in real-time exist. So far, optimized protocols have been prepared for stem cells, which are magnetically labeled by superparamagnetic iron oxide particles and tracked by magnetic resonance imaging (MRI). This cellular imaging technique, combined with contrast agents, has been widely used in in vitro studies, where grafted cells with efficient labeling did not exhibit impaired survival, proliferation, self-renewal or multipotency [94]. Cell integrity also depends on the progression of the disease and changes in functional status in ALS patients, which are measured using the Revised ALS Functional Rating Scale (ALSFRS-R)[93]. Additionally taken into account are also the estimated respiratory parameters and lifespan, as well as the records of a given number of assessed improvements in health as compared to control treatments.
4.1.
Clinical Application of NSCs for ALS therapy
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Due to the encouraging results of preclinical studies with NSC-based therapies, the US Food and Drug Administration approved a clinical trial using intra-spinal injection of human NSCs. In a phase I trial, 18 patients with ALS were monitored for up to 2.5 years after NSC transplantations. Twelve patients underwent unilateral or bilateral microinjections into the lumbar spinal cord, and six patients received unilateral microinjections into the cervical spinal cord. There were no observed major adverse events attributable to the surgery or cells [9596]. In November 2012, a phase II trial began, which is currently ongoing, but not recruiting new participants (ClinicalTrials.gov Identifier: NCT01730716). The aim of this study is to determine the efficiency, safety, toxicity, and maximal tolerated (safe) dose of NSCs for ALS therapy. The clinical trial, which started in July 2012 in Italy, was completed in December 2015 (ClinicalTrials.gov Identifier: NCT01640067). The phase I trial was performed on six ALS patients receiving unilateral or bilateral NSC microinjections into the lumbar spinal cord. The initial results demonstrated that, during the 18-month follow-up period after cell transplantations, surgical procedures did not cause an increase of disease progression. Moreover, two patients showed a transitory improvement in the ambulation sub-score on the ALS-FRS-R scale, another patient showed improvement in the Medical Research Council score for tibialis anterior muscles. Mazzini et al. (2015) described a safe cell strategy, which will allow for the treatment of larger patient populations in later-phases of ALS clinical studies. At the moment, the same research group in Italy is carrying out research on 12 ALS patients who underwent intra-spinal NSC injections into the cervical spinal cord [97]. 4.2.Clinical Application of BM-MSCs for ALS therapy Many cell-based clinical studies for ALS are underway using BM-MSCs. Most of them are in phase I/II, aimed to demonstrate the safety and feasibility of transplantation. In Israel, a phase I/II clinical trial was conducted on 15 patients with MS and 19 patients with ALS, confirming the safety and immunological effect of intrathecal and
17
intravenous delivery of MSCs. The 19 months of follow-up did not show any side effects in the course of the disease and indicated prolonged stabilization, or even improvements in certain patients, connected with a beneficial immunomodulatory effect [98]. A Korean research group has demonstrated that two repeated intrathecal autologous BM-MSCs injections (phase I trial) were safe and feasible for at least 12 months in 7 patients. No adverse effects were observed [99]. Apart from research concerning the safety of use of autologous MSC transplantation in a clinical trial in Turkey, an assessment of the secondary outcome in a study on 13 patients receiving MSC transplants into the cervical spinal cord showed promising effects. Nine of these 13 patients demonstrated motor improvement after cell delivery [100]. A research team in Spain evaluated 11 patients following intra-spinal MSC grafts. No serious adverse events were noted, and the number of MNs was greater in the areas of grafting [101]. In the above-mentioned cases, an alternative approach has also been used in which, instead of injecting MSCs, endogenous MSCs were mobilized in ALS patients by a granulocyte colony-stimulating factor. The trials carried out in Canada and Italy have shown the therapy to be safe, revealed mobilization of MSCs and confirmed anti-inflammatory responses in the spinal cord [102-103]. Another phase I clinical trial was carried out in Italy on 10 patients suffering from ALS. Autologous BM-MSCs were isolated, expanded in vitro, and suspended in autologous CSF. Subsequently, cells were delivered into the spinal cord at high thoracic level. The patients were subjected to observation, both before and after transplantation, by clinical, psychological, neuroradiological and neurophysiological evaluations. The study also indicated MSC transplantation to be safe and well-tolerated by ALS patients [104-107]. However, all ongoing clinical trials using BM-MSCs for the treatment of ALS patients are in phase I and/or II (Table 1). Therefore, more research is required in this direction.
18
5. Conclusions The limited number of clinical research indicates that NSCs and MSCs may bring be most effective positive effects in the treatment of ALS (Table 1). However, there are no clear guidelines for the optimal number of grafted cells, route of delivery, or the enrollment of patients for trials. It is also essential to have a certainty of the graft lifespan in order to reach permanent efficacy. The results of preclinical research indicate that transplanted human cells in animal models can be identified by immunohistochemistry using specific markers for human cells. A challenge for stem cell therapeutic approaches is also a need for the reliable tracking of grafted cells in clinical trials. Currently, although it is highly invasive and requires various tissue biopsies, histopathological evaluation is the “basic” method of monitoring transplanted cells. In recent years, however, advanced non-invasive cellular imaging techniques for visualizing engrafted cells in real time are being used more and more frequently. One of these advanced methods is MRI, which involves labeling cells with super paramagnetic iron oxide nanoparticles or with reporter genes and allows for an understanding of how the implanted cells migrate into or within the spinal cord, and their effect on degenerated motor neurons. Lately, a large portion of research is focused on stem cell-derived EVs, which mediate both immunomodulatory and neuroprotective effects, and might be engaged in the regeneration of damaged motor neurons. However, more studies on EVs need to be done to overcame problems connected with safety, manufacturing and availability, which would enable their use in clinical studies. It is therefore necessary to continue research and strive towards an effective cell-based therapy for the treatment of ALS.
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Acknowledgements This is supported by the National Centre for Research and Development Grant STRATEGMED1/234261/2NCBR/2014.
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Table 1. List of past and present clinical stem cell trials in ALS
Ordinal number
Type of stem cells
Specificity of cells
Application method
Status of trial
Trial institution/ sponsor and country
NCT number and duration period
1.
Neuronal Stem Cells (NSCs)
Human fetal spinal cord
Phase II, not verified status
2.
Mesenchymal Stem Cells (MSCs) Hematopoietic Stem Cells (HSCs)
Umbilical cord
Intraspinal Intrathecal Intrathecal
Neuralstem Inc., United States General Hospital of Chinese Armed Police Forces, China Hospital Universitario Dr. Jose E. Gonzalez, Mexico
NCT01730716 2013-2016 NCT01494480 2012-2015 NCT01933321 2012-2014
3.
4.
MSCs
Autologous, derived from patient bone marrow and mobilized to blood Autologous, derived from
Intrathecal
Phase II, patient’s recruitment Phase II/III, complete
Intrathecal
Phase I, patient’s recruitment
University of Warmia and Mazury, Poland
NCT02881489 2015-2018
Phase I, ongoing, but without patient’s recruitment Phase I/II, study withdrawn Phase I/II, not verified status Phase I, complete
Neuralstem Inc., United States
NCT01348451 2009-2016
patient bone marrow
5.
NSCs
Human fetal spinal cord
Spinal cord injections
6.
MSCs
Autologous
Intrathecal
7.
Mononuclear cells (MNCs)
8.
Stem Cells (SCs)
9.
MSCs
Autologous, derived from patient bone marrow Autologous, derived from patient adipose tissue Autologous, derived from patient adipose tissue
Intraspinal Intrathecal Intravenous Intracerebral Intrathecal
10.
MSCs
Autologous
Intrathecal
11.
MSCs
Allogeneic, derived from Wharton’s jelly umbilical cord
Intrathecal
Phase I, ongoing, but without patient’s recruitment Phase I, patient’s recruitment Phase I, patient’s recruitment
Alzahra Hospital, Iran
NCT02116634 2015-2017 Hospital Universitario NCT01254539 Virgen de la Arrixaca, Spain 2010-2015 China Medical University NCT02383654 Hospital, China 2015-2016 Anthony J. Windebank, NCT01609283 Mayo Clinic, United States 2012-2017 Hospital e Maternidade Dr. Christóvão da Gama, Brazil University of Warmia and Mazury, Poland
NCT02987413 2014-2017 NCT02881476 2015-2018
33
12.
Stem/progenitor cells
13.
MNCs
14.
Phase I, patient’s recruitment Phase I/II, complete
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow
Intrathecal
NSCs
Human fetal
Phase I, complete
15.
MNCs
16.
SCs
17.
MSCs
Intraventricular
18.
MSCs
Intrathecal
Phase I, patient’s recruitment Phase I, complete
19.
MNCs
Intrathecal
Phase I, complete
20.
SCs
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Adipose tissue is derived from healthy donors. Autologous, derived from patient adipose tissue Autologous, derived from patient bone marrow N/A
Spinal cord injections Intrathecal intramuscular Intrathecal
N/A
N/A, patient’s recruitment
21.
MNCs
Intramuscular
22.
Phase I, patient’s recruitment Phase I, study not open
23.
Neuronal Progenitor Cells releasing Glial cell-derived neurotrophic factor SCs
Autologous, derived from patient bone marrow Human fetal cortex
Intrathecal
Phase I/II, complete
24.
SCs
Intrathecal
Phase I, not verified status
25.
Glial Restricted Progenitor Cells (GRPs) MSCs
Autologous, derived from patient bone marrow Allogenic, derved from donor bone marrow N/A
Spinal cord injections Intraventricular
Phase I/II, study not open
26.
Autologous, derived from patient bone marrow
Intraspinal
Spinal cord injections
Phase II, complete Phase 1, study suspended
Phase I, complete
Pomeranian Medical University Szczecin, Poland Fundacion para la Formacion e Investigacion Sanitarias de la Region de Murcia, Spain Azienda Ospedaliera Santa Maria, Italy Neurogen Brain and Spine Institute, India TCA Cellular Therapy, United States Royan Institute, Iran Mayo Clinic, United States Neurogen Brain and Spine Institute, India University of Michigan, United States Red de Terapia Celula, Spain Cedars-Sinai Medical Center, United States
NCT02193893 2010-2017 NCT00855400 2007-2010
NCT01640067 2011-2015 NCT01984814 2008-2013 NCT01082653 2010-2014 NCT02492516 2014-2016 NCT01142856 2010-2011 NCT02242071 2008-2016 NCT02058732 2013-2019 NCT02286011 2014-2018 NCT02943850 2017-2019
Corestem, Inc., Korea
NCT01363401 2011-2013 Hanyang University Seoul NCT01758510 Hospital, Korea 2012-2017 Q Therapeutics, Inc., United NCT02478450 States 2017-2020 Royan Institute, Iran NCT01759797 2013-2014
34
Intraventricular
Phase I/II, patient’s recruitment Phase I, study withdrawn
University of Sao Paulo General Hospital, Brazil Royan Institute, Iran
Intrathecal
Phase I, complete
Royan Institute, Iran
Intravenous injections
Phase I/II, patient’s recruitment
Human Somatic Cells
N/A
N/A
Biopsy from patient skin
N/A
N/A
MSCs releasing Neurotrophic Factors (NTFs)
Autologous, derived from patient adipose tissue
Intramuscular Intrathecal
Phase II, complete
34.
MSCs releasing NTFs
intramuscular
Phase II, not verified status
35.
MSCs releasing NTFs
Autologous, derived from patient adipose tissue Autologous, derived from patient adipose tissue
Intramuscular intrathecal
Phase I/II, complete
Andalusian Initiative for Advanced Therapies Fundación Pública Andaluza Progreso y Salud, Spain Hadassah Medical NCT00801333 Organization, Israel 2008-2020 Institut Pasteur, France NCT01639391 2012-2014 Brainstorm NCT02017912 Cell Therapeutics, United 2014-2016 States Hadassah Medical NCT01777646 Organization, Israel 2012-2014 Hadassah Medical NCT01051882 Organization, Israel 2011-2013
27.
MSCs
28.
MSCs
29.
MSCs
30.
MSCs
31.
Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Autologous, derived from patient bone marrow Autologous, derived from patient adipose tissue
Inreathecal
Induced Pluripotent Stem Cells (iPSCs) IPSCs, Fibroblasts
33.
32.
NCT02917681 2016-2019 NCT01759784 2014-2017 NCT01771640 2013-2014 NCT02290886 2014-2021
35