Translational Research in Stem Cell Treatment of Neuromuscular Diseases

Neuromuscular diseases are a heterogeneous group of diseases that lead to significant disability in effected individuals. Pharmacological treatments failed to provide any significant improvement to date. Recently, the introduction of stem cells into the field of health sciences raised the hopes for a new treatment for neuromuscular diseases. In theory, stem cells, owing to their multilineage differentiation capacity, could differentiate into myofibers and neurons and replace the degenerated cells leading to recovery of the patients. Results obtained from the preclinical studies supported this theory. However, clinical trials with stem cells could not meet the expectations mainly because of early mortality, limited migration, and differentiation of the implanted cells. Modification of the stem cells before implantation, such as introduction of deficient genes or commitment to a precursor cell line provided little improvement. The biggest barrier to overcome for a successful of stem cell treatment, which also should be the focus of the future studies, is to increase the functional integration of the donor cells with the recipient tissues. Understanding the underlying pathogenic mechanisms of the neuromuscular diseases is essential to achieve this goal.


Introduction
e term neuromuscular diseases de�ne a wide range of conditions characterized by the weakness or wasting of the body muscles. Problems may primarily originate from the spinal cord, the peripheral nerves (neuropathies), the muscle �bers (myopathies), or the neuromuscular junction. Some of these diseases are hereditary, while others are acquired. e diagnosis is mainly done by clinical observation, electromyography, muscle biopsy, and in some instances molecular genetic studies. Some of the major types of neuromuscular diseases are amyotrophic lateral sclerosis (ALS), myasthenia gravis (MG), multiple sclerosis (MS), and muscular dystrophies (Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD)). Despite the long lasting research on the pathogenesis and the molecular mechanisms of neuromuscular diseases, no satisfactory treatment has been offered yet for these diseases [1].
Stem cell research is relatively new in the medical �eld but holds a great potential for the treatment of a variety of diseases that remained untreatable so far. Stem cells are believed to exist in all tissues in human body and may be totipotent, pluripotent, or multipotent, depending on tissue type. Neuromuscular degeneration itself seems to promote proliferation, migration, and transdifferentiation of autologous stem cells. Production of neurotropic and growth factors and stimulation of the regenerative processes by stem cells have been demonstrated in neurodegenerative diseases [2]. erefore, human skeletal muscle tissue and nerve tissue have a limited regeneration capacity via the muscle stem cells, also called satellite cells and nerve derived stem cells, respectively [3]. However, this regeneration capacity is not enough to reverse the pathological process in case of neuromuscular diseases. Following the discovery of multilineage plasticity of the stem cells that can be obtained in large numbers from easily accessible tissues, several attempts have been performed to treat neuromuscular diseases via the local or systemic injection of the stem cells. Despite the promising results obtained from in vitro studies, these attempts have yielded limited success in vivo as well as in clinical trials.

ISRN Stem Cells
is in part is due to the complexity of the microenvironment needed to ensure stem cell integration and function [3]. e aim of this paper is to summarize the translational research on the most common types of neuromuscular diseases.

Anatomy of Neuromuscular Unit
e formation of skeletal muscle begins during the fourth week of embryonic development by the rapid mitotic division of specialized mesodermal cells, termed myoblasts. By week nine of development, multinucleated skeletal muscle cells, termed muscle �bers, can be identi�ed. By month �ve, the muscle �bers begin to accumulate protein �laments important in muscle contraction. Muscle �bers aggregate into bundles as the fetus grows, and by birth myoblast activity, so the formation of new muscle �bers stops. Muscle �bers contain longitudinally arranged myo�laments, named actin and myosin. Muscle contraction on a subcellular level is a complex interaction of myo�laments coupled with the in�ux and e�ux of calcium ions following the excitation by nerve �bers. Each muscle �ber is bound to adjacent �bers to form bundles and accumulation of muscle bundles forms the muscle belly. Supporting connective tissue surrounding the individual �bers is named endomysium, the perimysium encloses the fascicles, and the epimysium surrounds the muscle belly [3].
Nerve �bers innervating the muscles originate from brain stem nuclei or the spinal cord and meet the muscle �bers in neuromuscular junctions. Neuromuscular junctions are composed of presynaptic nerve membrane, synaptic cle, and postsynaptic muscle membrane. Myelinated motor nerve �bers enter their target muscles and they divide into 2�-1�� unmyelinated terminal �bers, each of which innervates a single muscle �ber. e terminal �ber from a motor axon and the muscle �ber it innervates are called a motor unit. e terminal nerve �bers contain the neurotransmitter acetylcholine (ACh) stored in synaptic vesicles. When the nerve �bers are stimulated by an action potential, ACh is released into the synaptic cle and binds to the ACh receptors on the postsynaptic muscle membrane initiating the muscle contraction [6].

Stem Cells
Stem cells can be classi�ed according to the way that they are derived and the source tissues. Embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), haemopoietic stem cells (HSCs), and induced pluripotent stem (iPS) cells are the major types of stem cells. Each one of these stem cell types possesses certain advantages and disadvantages which should be weighed carefully before the desired applications.
ESCs cells are derived from the inner cell mass of a developing blastocyst and are pluripotent, possessing the capacity to give rise to all 3 germ layers. Concerns about in vivo tumor formation and ethical concerns regarding their harvest have thus far restricted the use of ESCs [3]. MSCs and HSCs are multipotent, self-renewing cells derived from adult tissues that can form a number of cells or tissues that are usually restricted to a particular germ layer. e major advantages of the MSCs and HSCs over ES cells are the ease of harvest and lack of immunoreactivity since they are derived from autologous sources. However, they may be less desirable for the treatment of genetic diseases since they may possess the same genetic predisposition to the disease. For example, MSCs derived from ALS patients exhibit diminished growth and differentiation capacity [7].
Most recent advancement in the �eld of stem cell research is the development of iPS cells [8]. iPS cells are reprogrammed �broblasts that are transfected by selected transcription factors delivered by vector-, virus-, protein-, or RNA-mediated approaches. Original protocol utilized transcription factors Oct 3/4, Klf, Sox2, and c-Myc; however multiple research groups have now accomplished successful reprogramming of �broblasts using various combinations of factors [9][10][11][12]. ese cells are not only an option for disease modeling but also provide a novel source for autologous cellular therapies [7].
Stem cells are used in two different ways in neurodegenerative diseases: direct differentiation and regeneration or paracrine anti-in�ammatory activity. Moreover, stem cells might become eventually carriers of pharmacological and gene treatments [2].

Amyotrophic Lateral Sclerosis
ALS is a neurodegenerative disease caused by the selective loss of both spinal and upper motor neurons [13]. It is the most common motor neuron disease in adults and usually diagnosed in the sixth decade of life. Initial presentation is limb weakness which progresses gradually to generalized muscle atrophy and paralysis. Respiratory muscle involvement leads to death usually within � years. e de�nitive treatment of the disease is the replacement of the lost motor neurons. Many pharmacologic agents have been tried for the treatment of ALS but none of them proved to be effective up to date.
Stem cells have been extensively experimented for the treatment of ALS in animal models and some provided signi�cant bene�t in preclinical level (Table 1). e types of stem cells that have been tested include neural stem and progenitor cells (NSCs, NPC), umbilical cord blood stem cells (UCBCs), bone marrow stem cells (BMCs), human glial restricted progenitors (hGRPs), embryonal stem (ES) cells, glial restricted progenitors (GRPs), and olfactory bulb neural progenitor cells (OB-NPCs). ere are around ten clinical trials reported so far placing the amyotrophic lateral sclerosis to the top of the list of clinical trials for neurodegenerative disorders ( Table 2). Even though some of these trials reported a limited success in terms of prolonged life span and functional improvement, the desired level of success has not been reached yet. Mazzini  study [14]. ey later on published larger patient series along with the long-term follow-up results [2,15,16], and in their �nal paper in 2012 they stated that BMC application does not improve the symptoms of ALS in the long term, however, stabilizes the symptoms of the disease thus prolonging the life span of the patients [17]. On the other hand, Nefussy et al. could not detect any bene�t of stem cell treatment in case of ALS, but it should be mentioned that they mobilized the BMCs via injection of granulocyte colony stimulating factor (G-CSF) but did not carry out direct injection of the cells [18]. A common �nding of these clinical trials was the lack of major side effects of intraspinal/intracerebral cell injection that makes the clinicians more comfortable in continuing their efforts for the re�nement of the stem cell therapies for ALS [14,15,19,20]. erefore, more clinical trials are on the way and there are nine more in various stages listed in the NIH database for clinical trials.

Multiple Sclerosis
MS is a chronic in�ammatory demyelinating disease of the central nervous system that leads to cumulative and irreversible damage. MS is characterized by self-reactive lymphocytes and demyelination. erefore, any method for the treatment of MS, to be effective, should reverse the demyelination as well as suppressing self-reacting lymphocytes. Available drug therapies are only partially effective in these terms. However, stem cells, considering their immunosuppressive effects and multilineage differentiation capacity, may be applicable for meeting some of these goals. Preclinical data and ongoing clinical trials suggest that selected patients may respond positively to autologous stem cell transplantation.
But it is still uncertain if adult stem cells can repair existing neurological de�cits in patients with MS [21]. e stem cell types that have been used for the treatment of MS in animal models were BMCs, iPS, NSCs, NPCs, HSCs, ESCs, adipose-derived stem cells (ASCs), embryonal carcinoma cells, bone marrow transplantation (BMT), Schwann cells (SCs), Schwann cell precursors (SCPs), and oligodendrocyte progenitor (OCP) cells. It was Bonilla et al. who �rst used stem cells for the treatment of MS [22]. ey have injected BM-derived HSCs into the mouse brain and obtained encouraging results. Numerous studies have followed and they are listed in Table 3. Oka et al. used primates for their experiments and injected monkey NPCs into the central nervous system of the monkeys to treat MS [4] ( Figure 1). ey have seen signi�cant number of myelinating �bers in the transplantation zone. In another primate study it was reported that transplanted human NPCs decreased disability and increased survival of the animals [23]. e results of these two experiments were important in terms of the proximity of the animal model to human beings. e potential of iPS cells to differentiate into functional OPCs, as documented recently, is another milestone development that would ameliorate the stem cell donor site issues [24]. ere is only one controversial study in the literature published by Reekmans et al. in which mouse NSCs were transplanted to a mouse MS model but no therapeutic effect or improvement in the disease state could be detected [25].
Clinical trials on the treatment of MS with stem cells are listed in Table 4. In the standard protocol, patients are �rst treated with a combination of immunosuppressive agents to eliminate all lymphocytes including self-reactive lymphocytes. Myeloablative treatment is followed by stem Mouse NPCs Mouse Decreased in�ammation in brain but no effect in spinal cord lesions [87] Mouse NPCs Mouse Reduced migration of transplanted cells in chronic disease [88] Mouse NPCs Mouse Enhanced remyelination [84] Mouse NPCs Mouse NPC differentiated into oligodendrocytes in vivo in long term [87] Mouse Triggering receptor expressed on myeloid cells 2, * * interferon-, * * * brain-derived neurotrophic factor. cell transplantation. However, direct intrathecal injection of stem cells as well as nonmyeloablative stem cell transplantation has also been tried with favorable results [26,27]. e largest series published so far includes 183 patients that are recorded in the database of the European Blood and Marrow Transplantation Group [28]. All these patients were treated with HSCs, and overall transplant-related mortality was 5.3% with an improvement or stabilization of neurological conditions in 63% of patients during a median followup of 41.7 months. e only reported serious side effect of stem cell treatment of MS was the Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder [29]. In summary, both myeloablative or nonmyeloablative stem cell applications seem to be safe and both provide a certain level of clinical bene�t to the patients.

Muscular Dystrophies
DMD is characterized by a progressive degeneration of the whole body musculature due to a de�cit in the dystrophin gene. Dystrophin is a large protein of skeletal muscle tissue that is expressed under the sarcolemma and contributes to the stability of the giant syncytial muscle �bers. Natural course of the disease is con�nement to wheelchairs around the age of 12 and death ensues in the later stages due to cardiomyopathy. During the course of the disease, effected skeletal muscle tissue regenerates to a limited extend through the activation of satellite cells but multiple cycles of degenerationregeneration eventually lead to exhaustion of the myogenic reservoir. Replenishment of the myogenic reservoir is the principle of the stem cell transplantation to the skeletal muscle in muscular dystrophies. BMD is very similar to DMD except that it gets worse at a much slower rate and it is less common [30]. DMD and BMD are the most extensively studied diseases in the group of neuromuscular disorders. erefore it would be overwhelming to cite all the experimental papers on treatment of muscular dystrophies in this paper. Instead, a general summary of the preclinical studies is given in Table  5. e most commonly used cell type in preclinical studies was myoblasts that originate from myogenic progenitors (MPCs), and the main MPCs in skeletal muscles are satellite cells. ese normally quiescent cells are located beneath the muscle �ber basal lamina, and upon activation they either differentiate into committed progenitors (myoblasts) or self-renew by asymmetric division [3,30,31]. However, the �rst experimental report by Ferrari  and reported that the graed cells fused only with the recipient �bers at the injection site but did not migrate for so long distances limiting the success of the treatment [33]. Gene delivery methods using stem cells as vehicles proved some improvement [5,[34][35][36][37]. Tedesco et al. transplanted genetically corrected mesoangioblasts in SCID/mdx mice intramuscularly [5]. ey have successfully documented the dystrophin production and amelioration of morphological defects in tibialis anterior muscle of SCID/mdx mice following cell transplantation (Figure 2). While most of the other studies documented the limited success of stem cell treatment in muscular dystrophies, some others reported no bene�t [38][39][40].
Despite the high number of preclinical studies and encouraging data, clinical trials have been hampered by poor survival and limited migratory ability of the cells (Table 6). Neumeyer et al. reported no positive effect of myoblast transplantation into the tibialis anterior muscles of the patients with BMD [41]. Skuk et al. could detect fusion of the donor and recipient muscle �bers and subsequent synthesis of donor dystrophin following MPC allotransplantation; however this observation was limited to the injection sites [42]. Zhang et al. reported similar results following the transplantation of allogeneic human UCBCs [43]. Oxidative stress, fusion inability, and some administration methodologies are the suspected causes of poor cell survival following transplantation. Intramuscular administration was the preferred method of cellular delivery in most of the clinical studies since many cell types are not able to cross the endothelial barriers of the skeletal muscle tissue. e migration of the injected cells was generally restricted to short distances from the injection site. e resultant large intramuscular pockets of cells subsequently lead to cell death due to inefficient nutrient supply in vivo. Currently, there is only one ongoing clinical trial on stem cell treatment of muscular dystrophies in the NIH database. In this study the researchers aim to treat DMD by human UCBC transplantation.

Myasthenia Gravis
MG is an Ach receptor antibody-mediated autoimmune neuromuscular disease. e pathogenesis of MG involves the hyperactivity of T lymphocytes, activation of complement system, de�ciency of immunomodulation, and impairment of immune homeostasis.
MSC has got the potential to correct impaired immune homeostasis in diseases like MG because they express intercellular adhesion molecule (ICAM)-1, ICAM-2, lymphocyte function-associated antigen (�FA) 3, �bronectin, lamin, and collagen, which are involved in the process of immune reaction. Moreover MSCs can modulate the T lymphocyte function via direct physical contact through the cell adhesion molecules [44,45]. However, there are a just a few experimental studies on the application of stem cells for the treatment of MG. e �rst study was published by �u et al. [44]. In this study BMCs were applied intravenously to a mouse model of MG. e authors noted a signi�cant decrease in the circulating levels of Ach receptor IgG antibody and a signi�cant functional improvement. e same group injected human UCBCs to mouse model of MG in another experiment and obtained similar results [46]. Sheng et al. injected granulocyte macrophage colony stimulating factor (GM-CSF) to a mouse model of MG and noted a decreased immune response against the Ach receptors through the mobilization of tolerogenic precursor cells [47]. e results  No local or systemic side effects were observed. Treated patients had an increased ratio of capillary per muscle �bers with a switch from slow to fast myosin-positive myo�bers. [187] UCBCs 100 days Restoration of the dystrophin in muscles, and improvement of the locomotive function. [43] of these experiments were encouraging in terms of clinical application, and a phase I clinical trial is already on the way (ClinicalTrials.gov identi�er: NCT0042448�). e purpose of this study was stated as to assess the toxicity/feasibility of autologous hematopoietic stem cell transplantation for refractory MG cases in the NIH database. Several other studies are required to assess the feasibility of stem cell therapy for MG, and the concerns about the safety of the stem cell application in case of MG should be further investigated.

Discussion and Conclusions
Stem cells are the regenerative reservoirs of the body. ey are located in niches in the tissues and become active in case of tissue injury to replace the degenerated cells. ere are two possible mechanisms for the therapeutic effects of the stem cells: differentiation into different cell types or paracrine secretion. Some neuromuscular diseases like MS, ALS, and MG have got an in�ammatory component, and in this case anti-in�ammatory and immunomodulatory effects of stem cells could provide a treatment. In case of muscular dystrophies, the mechanism is the degeneration of muscle �bers rather than autoimmune destruction so the differentiation capacity of the stem cells comes forward. Currently there is a gap between animal and human applications of stem cells in neuromuscular disorders so as the promising results obtained in animal studies have not been reproduced in clinical trials yet. e reasons for this failure can be summarized as limited differentiation of stem cells into required cell types, short survival times of transplanted cells, and lack of widespread effect due to the limited migration of the cells away from the injection site. In case of neuromuscular diseases, the preferred way for the delivery of the cells should be direct injection to the target tissues since the biological barriers may limit the migration of stem cells to the target tissues thus decreasing the efficacy of the treatment. Nevertheless lack of long distance migration of the stem cells in the tissues is still a major obstacle in front of cellular therapies as mentioned above. Ideas like corrective gene delivery via stem cells are theoretically brilliant, however, despite some improvement, they have not reached the desired clinical effectiveness yet. One single common result of stem cell trials is the safety of the procedure. erefore further research can safely be carried out to overcome the current obstacles. Moreover, introduction of new stem cell types such as iPS cells will help to overcome donor site and ethical issues and increase the availability of stem cells.
In conclusion, stem cells still hold a great promise for the treatment of traditionally untreatable diseases. Continuing research will enable the clinicians to understand the biological processes of the neuromuscular diseases better and allow the modi�cation of current treatment protocols accordingly. However the expectations from clinical applications should be kept realistic, and more importantly best effort should be done to prevent the worsening of the pathologies by cellular treatments.