Clinical relevance of stem cell therapies in amyotrophic lateral sclerosis
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.136895
Source of Support: None, Conflict of Interest: None
Amyotrophic lateral sclerosis (ALS), characterized by the progressive loss of both upper and lower motor neurons, is a fatal neurodegenerative disorder. This disease is often accompanied by a tremendous physical and emotional burden not only for the patients, but also for their families and friends as well. There is no clinically relevant treatment available for ALS. To date, only one Food and Drug Administration (FDA)-approved drug, Riluzole, licensed 18 years ago, has been proven to marginally prolong patients' survival without improving the quality of their lives. Because of the lack of an effective drug treatment and the promising outcomes from several preclinical studies, researchers have highlighted this disease as a suitable candidate for stem cell therapy. This review article highlights the finding of key preclinical studies that present a rationale for the use of different types of stem cells for the treatment of ALS, and the most recent updates on the stem cell-based ALS clinical trials around the world.
Keywords: Amyotrophic lateral sclerosis, cell transplantation, clinical trials, motor neuron degeneration, regenerative medicine
Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a rapidly progressive neurodegenerative disorder characterized by rapid and unremitting degeneration of both upper and lower motor neurons. The loss of motor neurons leads to muscle atrophy and fasciculation, marked progressive weakness, spasticity, dysarthria, dysphagia, dyspnea, and, ultimately, death. Most patients with ALS die within three to five years of symptom onset, usually due to respiratory failure. The incidence is around two to three cases per 100,000 of the general population annually, and the prevalence is around four to six per 100,000.  ALS is a heterogeneous disease and both genetic and environmental factors are responsible for the condition. The majority of cases are sporadic, and approximately 10% are the familial form of ALS (fALS).  Predominantly, fALS is inherited in an autosomal dominant manner and 12 to 13% of these cases are associated with missense mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1)  In addition to genetic factors, environmental factors, such as the use of tobacco, toxins, toxic metals and solvents, pesticides, and strenuous physical labor may also contribute to the disease. Despite the identification of several risk factors for ALS, in most cases, the etiology remains unidentified. Similarly, the mechanisms responsible for disease onset and progression in ALS are also largely unknown. Histological analysis of ALS tissue samples have shown that motor neurons in the oculomotor (CN III), trochlear (CN IV), and abducens (CN VI) nerves, and Onuf's nuclei are unaffected , , whereas motor neurons in the brain and spinal cord degenerate in specific patterns for each patient.  The proposed pathomechanisms for motor neuron degeneration in ALS, albeit not fully understood, include protein aggregation, glutamate-mediated excitotoxicity, mitochondrial dysfunction, oxidative stress, impaired axonal transportation, altered glial cell function, and insufficient neurotrophic factors. 
Currently, there is no clinically relevant treatment available for ALS. Several treatment modalities have been attempted in the past, ranging from the use of drugs with anti-inflammatory, anti-oxidative, and anti-apoptotic properties, drugs providing trophic factors, as well as glutamate receptor blockers, all with a lack of robust results in clinical trials.  There is only one Food and Drug Administration (FDA)-approved drug for ALS treatment, Riluzole, which is an anti-glutamate agent with an unclear mechanism of action that marginally extends the life-span of patients by two-to-three months.  In the absence of any effective treatment, ALS patients are still in desperate need of new therapies.
Considering the complexity of the disease and the poorly understood pathomechanisms, the development of a therapy that can maintain or restore motor neuron function would provide the most comprehensive approach to treating ALS. In past decade, several preclinical studies have shown promising outcomes using stem cells in ALS transgenic animal models. In 2009, the FDA approved the first phase I safety trial of direct intraspinal transplantation of neural stem cells (NSCs) into patients with ALS. The procedure was found safe, feasible, and well tolerated by patients with no long-term complications associated with the surgical procedure or cell toxicity. , The public clinical trials database, www.clinicaltrials.gov, currently lists several clinical trials at different stages around the world using stem cell-based therapies for ALS. This review article highlights the outcomes of key preclinical studies using different types of stem cells and discusses the most recent updates on stem cell-based clinical trials for ALS.
The development of relevant therapies for ALS has proven to be challenging particularly because of the insidious, neurodegenerative course of the disease. The manifestation of symptoms in ALS is very late, which means that by the time the disease is diagnosed, a very large pool of motor neurons has either already been lost or is rapidly marching toward degeneration. Thus, in order to treat ALS, degenerating motor neurons ultimately need to be protected, regenerated, or replaced. Stem cell therapy is an innovative approach for ALS treatment given the ability of stem cells to differentiate into multiple neuronal lineages. After systemic or local transplantation, stem cells have the ability to migrate to pathologic sites in the body and produce a therapeutic effect.  Transplanted stem cells could replace dead motor neurons or provide protection to surviving host motor neurons. Preclinical studies have demonstrated that intraspinally transplanted stem cell-derived motor neurons could indeed integrate, receive, and make synapses, extend axons, form neuromuscular junctions, and recapitulate a neural network.  Alternatively, transplanted stem cells could provide environmental enrichment to support endogenous dying motor neurons by producing neurotrophic factors, scavenging toxic molecules, or creating auxiliary neural networks around affected areas. , Past studies have presented a tremendous amount of evidence about the types of stem cells that are likely to offer therapeutic benefits in ALS, and, based on that evidence, several clinical trials are currently underway.
Different types of stem cells have been tested for therapy in ALS [[Figure 1], reproduced from Ref. 93, with permission]. Below is a description of the types of stem cells that have shown promising clinical outcomes.
Human umbilical cord blood derived-cells
The human umbilical cord blood (hUCB) is a very rich source of multipotent stem cells. The hUCBCs are capable of developing into cells of various tissue lineages, including neural cells. These cells display very low immunogenicity,  which may limit the incidence and severity of graft-versus-host diseases after transplantations. In neurodegenerative conditions like stroke, traumatic brain injury, spinal cord injury, and Alzheimer's disease, engraftment of a mononuclear cell fraction derived from hUCB has been found effective.  The hUCBCs secrete a number of growth factors, and thus, may contribute to a regenerative microenvironment for the neurons.  However, there is no detailed knowledge about the precise mode of action.
Preclinical studies using hUCBCs in ALS
In ALS mice, hUCBCs were found to have bystander trophic effects on motor neurons. As shown in a study, in presymptomatic ALS mice, genetically modified hUCBCs [overexpressing human vascular endothelial growth factor (VEGF) and human fibroblast growth factor (FGF)], when retro-orbitaly injected, transformed into astrocytes and served as a source of growth factors to enhance the survival of motor neurons.  In several other studies, hUCBC treatment was found to delay disease progression, improve motor function and neuromuscular transmission, attenuate astrogliosis, and improve survival in ALS mice. ,, However, the efficacy of hUCBC treatment greatly depends on the number of cells transplanted. For example, retroocular injection of hUCBCs into the venous system of presymptomatic irradiated ALS mice at relatively large doses extended the lifespan of the mice in a dose-dependent manner. , Similarly, in another study, multiple intravenous injections of hUCBCs were required to protect motor neurons from inflammatory effectors in ALS mice. 
Although clinical application of these cells in ALS is not yet imminent, hUCB-based therapy may have the potential for translation to clinic. Currently, data available on www.clinicaltrials.gov show only one ALS clinical trial using hUCBCs (ClinicalTrials.gov Identifier: NCT01494480) at the General Hospital of the Chinese Armed Police.
Embryonic stem cells
Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of an embryo at the blastocyst stage. These cells are distinguished by their ability to differentiate into derivatives of all three germ layers (endoderm, mesoderm, and ectoderm) and by their ability to provide a seemingly unlimited supply of specific cell types. By using different cell-signaling molecules, ESCs can be differentiated into motor neurons and interneurons,  dopamine neurons,  astrocytes,  oligodendrocytes,  and microglia. 
Preclinical studies using ESCs in ALS
One potential strategy for ALS treatment is to replace the endogenous damaged neurons with fresh exogenous motor neurons. It is feasible to direct the differentiation of ESCs into spinal motor neurons using cell-signaling molecules  and then engraft these neurons for replacement of degenerated neurons. Past studies have shown that motor neurons generated from ESCs can maintain molecular and functional characteristics, and show functional engraftment after transplantation into the spinal cords of developing chicks and adult rodents with motor neuron degeneration. ,, In one study, Harper et al., reported that in a rat model of virus-mediated acute motor neuron death, ESC-derived motor neurons could extend axons into the ventral roots when animals were co-infused with dibutyryl cAMP,  a membrane-permeable cAMP analog, which enables mature neurons to extend axons in the presence of myelin inhibitors. In ALS transgenic rats, transplantation of mouse ESC-derived motor neurons in the lumbar spinal cord region transiently improved the functional outcome, but there was no long-term effect on the survival of rats and poor graft survival was observed.  A hostile microenvironment in the ALS spinal cord could be responsible for the poor graft survival. For the successful treatment of ALS, engrafted motor neurons should not only survive, but also be able to send axons through inhibitory white matter and direct axons over long distances to the target muscles in order to restore neuromuscular function. More studies are needed to demonstrate these steps in ALS animal models.
Currently, no clinical trial is underway using ESCs in ALS patients. Ethical concern is one of the reasons that limit the use of these cells in the clinic. A thorough evaluation of the long-term risks associated with ESC-based therapy, such as tumor formation, unwanted immune responses, and the transmission of adventitious agents (bacterial, viral, fungal, or prion pathogens), must be a pre-requisite step before widespread clinical application is a reality.
Induced pluripotent stem cells
Induced pluripotent stem cells (iPSCs) are artificially derived cells from adult somatic cells and share many properties with ESCs, with no ethical concerns. These cells can be easily generated from fibroblasts, , and further differentiated in vitro in specific cell types. Recently, motor neurons and neural progenitors have been successfully generated from both ALS patients and mice iPSCs, ,,, and studies are now underway to examine their ability to migrate and successfully modify symptoms of disease.
Preclinical studies using iPSCs in ALS
To describe the in vivo fate of transplanted iPSCs in the ALS microenvironment, Popescu et al., transplanted iPSC-derived neural progenitors into the spinal cord of wild-type and transgenic rats carrying a human mutated SOD1(G93A) gene. They reported that iPSC-derived neural progenitors efficiently engrafted in the spinal cord and survived in great numbers. Over time, the transplanted cells differentiated into cells displaying a neuronal phenotype. However, this study did not evaluate axonal outgrowth, synaptogenesis of the engrafted cells or the effect of engraftment on functional outcomes, such as motor deficits, or a delay in disease onset and survival.  Recently, Nizzardo et al., demonstrated that intravenous and intrathecal transplantation of human iPSC-derived neural progenitors with high aldehyde dehydrogenase (ADH) activity and integrin VLA4 positivity, could significantly improve the neuromuscular function and the motor unit pathologically in ALS transgenic mice. The engraftment also improved the disease phenotype and survival of ALS mice. Interestingly, the authors reported a better therapeutic effect when the cells were administered systemically compared to intrathecally. 
Epigenetic re-programming of adult human somatic cells into iPSCs has enabled the generation of disease models of neurodegenerative disorders for the study of disease mechanisms. These cellular models can also been used as a platform for drug screening. The usefulness of iPSCs to project as a model for different diseases has been extensively reviewed by Cherry and Daley.  The iPSCs have been successfully used to model ALS, investigate sporadic and familial ALS pathogenesis, , and to perform drug screening for ALS using patient-specific iPSCs.  The National Institute for Neurological Disorders and Stroke (NINDS) Repository at the Coriell Institute for Medical Research has generated an open-access collection of fibroblast lines from patients carrying mutations that are linked to different neurological diseases. Any research group can request these cell lines for in vitro disease modeling. Currently, 18 mutation-defined cell lines from ALS patients are available upon request and this collection is continually expanding. An up-to-date list of cell lines can be found at: http://ccr.coriell.org/sections/collections/NINDS/FibroSubcollList.aspx?SsId = 10 and PgId = 681.
For regenerative purposes, iPSCs, unlike ESCs, can be generated and administered autologously, avoiding allogeneic immune rejection, and there are no ethical and fewer regulatory barriers to clinical development. Despite these benefits, a series of challenges remain for the clinical use of iPSCs. For example, the aberrant expression of re-programming factors, clonal selection, and prolonged in vitro culture are potential pathways for causing genomic alterations in iPSCs. Also, like ESC-based cell based therapies, long-term risks and safety must be evaluated before iPSC-based cell therapies are used in ALS patients.
Mesenchymal stem cells
Mesenchymal stem cells ( MSCs) are multipotent cells that possess self-renewal ability and can be differentiated into mesoderm lineages, such as chondrocytes, osteocytes, and adipocytes.  MSCs are believed to exist in almost all tissues. Although bone marrow is the best characterized source of MSCs, they can be easily isolated from cord blood, Wharton's jelly, placenta, adipose tissue, fetal liver, muscle, and lung. MSCs exhibit a multitude of positive effects when engrafted, including the release of pro-survival trophic and immunomodulatory factors and, when administered systemically, always migrate to the damaged tissue sites with inflammation. , MSCs have been used in the clinic for approximately 10 years and have been found effective in various neurological conditions. ,,,, Because of the inflammation directed-homing, and the ability to release pro-survival and immunomodulatory factors, MSCs are attractive therapeutic cells as modifiers of the ALS-specific inflammatory and excitotoxic microenvironment in the spinal cord.
Preclinical studies using MSCs in ALS
Given the fact that MSCs can deliver neurotrophic, anti-inflammatory, and immunomodulatory molecules, they are the most widely used cells for therapy in ALS. In several animal studies, MSCs have been shown to effectively ameliorate the clinical and pathological features of ALS. Different routes of MSCs delivery have shown a different range of therapeutic effects in ALS animals. Intraparenchymal transplantation of human MSCs into the lumbar spinal cord of asymptomatic ALS mice was found to delay motor neuron death, improve motor function, and reduce neuroinflammation.  Similar therapeutic effects were observed when cells were delivered intravenously into irradiated asymptomatic ALS mice.  In one study, Uccelli and colleagues showed that intravenously transplanted MSCs in ALS mice improved survival and motor functions. They also observed a reduced accumulation of ubiquitin agglomerates, and no changes in the number of choline acetyltransferase (ChAT) and glutamate transport type 1-positive cells in the spinal cord of MSC-treated ALS mice compared to saline-injected controls.  In symptomatic ALS mice, the systemic administration of adipose-derived MSCs significantly delayed motor deterioration for 4-6 weeks, and preserved the lumbar motor neurons, as well as the unregulated levels of glial-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) in the spinal cord.  In another study, it was found that combined intraparenchymal and intravenous engraftment of MSCs had a beneficial and possibly synergistic effect on the lifespan of ALS animals.  Murine MSCs, intrathecally administered into symptomatic ALS mice, not only improved the clinical outcome, but were also able to migrate to the ventral gray matter of the spinal cord and differentiate into astrocytes.  However, in another study, when MSCs that were isolated from the bone marrow of an ALS patient and intrathecally transplanted into the cerebellomedullary cistern of another symptomatic ALS animals, showed a dose-dependent efficacy.  In contrast to the beneficial outcomes of other routes of cell deliveries, intracerebroventricular injection of MSCs via the fourth cerebral ventricle was able to prolong the survival of ALS mice, but only in females, and had no effect on the survival of motor neurons or on neuroinflammation.  However, MSCs expressing glucagon-like peptide-1 (GLP-1), a peptide with antioxidant properties, when transplanted via intracerebroventricular injection in ALS mice, significantly delayed disease onset, improved survival, and suppressed neuroinflammation.  Intramuscular delivery of MSCs expressing the increased levels of GDNF were found to ameliorate motor neuron loss within the spinal cord where it connects with the limb muscles receiving transplants, and increased the lifespan of ALS rats.  In another study, MSCs genetically modified to release GDNF or VEGF, when bilaterally injected into the tibialis anterior, the forelimb triceps brachii, and the long muscles of the dorsal trunk muscles, prolonged survival and slowed the loss of motor function. Interestingly, the combined delivery of these neurotrophic factors in the muscles of ALS animals also showed a strong synergistic effect. 
As defined by European regulations on advance therapies, MSCs are considered to be an advanced therapy medicinal product (European Regulation #1394/2007). Early clinical investigations indicated that intra-spinal transplantation of autologous MSCs is feasible in ALS patients. [Figure 2] (Reproduced from Ref. 94, with permission) shows a schematic of targeted injection of stem cells into the spinal cord. Two open-label pilot studies from Italy demonstrated the safety and feasibility of intraparenchymal transplantation of bone marrow-derived MSCs into the thoracic spinal cord (T4-T5 and T5-T6) of 19 ALS patients. The cell transplantation was found to be safe and well tolerated by the patients, with no major adverse events, such as respiratory failure or death in any of the patients. ,,, The patients were followed for nine years and follow-up brain MRI revealed no structural changes and no deterioration in the psychosocial status of patients.  Although the study was limited by a lack of any pathological evaluations of transplanted cells, it represented a first step towards an understanding of the safety profile of intraspinal cell engraftment. In another clinical trial conducted at the University of Murcia, Spain, 11 ALS patients were intraspinally transplanted with autologous bone marrow mononuclear cells into the thoracic spinal cord (T3-T4). Similar to the above-mentioned study, no major adverse events were observed in the patients. However, 43 non-severe events were noted during the two years of follow-up. Four patients died on days 359, 378, 808, and 1,058 post-transplant for reasons unrelated to the procedure, and the pathological analysis of the spinal cord showed the presence of a greater number of motor neurons in the treated segments, as well as evidence of neurotrophism, compared to the untreated segments. , Similarly, a clinical trial in Turkey has shown encouraging secondary outcomes after 13 sporadic ALS patients were transplanted with bone marrow-hematopoietic progenitor stem cells into the cervical spinal cord (C1-C2). During the one-year of follow-up after cell transplantation, nine patients showed improvement compared to their pre-operative status, confirmed by electroneuromyography.  Because both upper and lower motor neurons are affected in ALS, the delivery of cells to regions of the motor cortex could also be another approach to the treatment of ALS. A pilot study in Mexico examining the safety and feasibility of intracranial (frontal motor cortex) delivery of MSCs in ALS patients found that the survival rate in treated patients was significantly higher than that in untreated patients. , However, the lack of baseline observations, such as follow-up MRI and measures of upper motor neuron function, makes it difficult to interpret whether the therapeutic effects seen in this study were attributable to the transplantation of the cells themselves. Intrathecal and intravenous delivery of autologous MSCs in ALS patients was also found to be safe in clinical trials. A clinical study from India successfully demonstrated the safety and feasibility of intrathecal transplantation of autologous bone marrow-derived stem cells in ten patients with ALS.  Systemic delivery of MSCs has also been shown to be efficacious. In a phase 1/2 open-safety clinical trial from Israel in 19 ALS patients, an immediate immunomodulatory effect was observed after intravenous delivery of MSCs in five patients (blood samples of only five patients of 19 were analyzed). Overall, no major adverse effects were reported during follow-up (≤25 months) and the whole procedure was found to be safe and feasible.  Three different clinical studies used an innovative approach in which, instead of transplanting MSCs, mobilization of endogenous MSCs was transiently increased using granulocyte-colony stimulating factor (G-CSF) in ALS patients. No adverse events were noted in any of the three conducted studies. ,, Intramuscular grafting has also been considered for cell delivery in ALS patients. An Israel-based company, BrainStorm Cell Therapeutics, Inc., has developed a cell type called 'NurOwn' for the treatment of ALS. NurOwn cells are stimulated bone marrow-derived autologous MSCs that secret neurotrophic factors. In a study of these cells, a 75-year old man diagnosed with 'myasthenia gravis' and ALS was treated with NurOwn cells. One month after receiving both intrathecal and intramuscular injections of the cells, the patient demonstrated significant improvement in cognition, speech, and muscle power.  Pending FDA approval, a phase II clinical trial using NurOwn cells is expected to start in 2014 (ClinicalTrials.gov Identifier: NCT02017912).
Neural stem cells
Neural stem cells (NSCs) are defined as a population of self-renewing multipotent progenitor cells present in the embryonic and adult central nervous system.  The NSCs have the ability to symmetrically self-renew and differentiate into neurons, astrocytes, and oligodendrocytes through asymmetrical fate-committed division. Based on previous evidence, it seems that NSCs might result not only in the regeneration of lost tissue, but also have the ability to improve functional outcomes through bystander mechanisms, such as neurotrophism ,, and immunosuppressive effect. ,, Transplantation of NSCs in various animal models of neurodegenerative disease has demonstrated promising outcomes and it is hoped that NSCs will become a promising therapy for the treatment of various neurological disorders. 
Preclinical studies using NSCs in ALS
In several preclinical studies, the therapeutic potential of both mouse and human NSCs has been tested in ALS animals. Two studies have examined the effect of human NSCs, isolated from the spinal cord of an 8-week-old fetus, intraspinally transplanted into the lumbar region of immunosuppressed presymptomatic ALS rats. , After transplantation, NSCs have extensively differentiated into neurons and formed synaptic contacts with host nerve cells, delayed disease onset and progression, and extended the survival of the animals by more than 10 days.  The combination of CD4 antibodies with immunosuppressive drugs was more effective and resulted in a further prolongation of animal survival. In another study, intraspinally engrafted human NSCs showed an advanced degree of structural integration, via functional synapses, into the segmental motor circuitry of ALS rats.  Hefferan et al., found that grafting of human NSCs into the lumbar spinal cord of ALS rats protected motor neurons in the vicinity of grafted cells and provided transient functional improvement, but was not able to protect motor neurons distal from the grafted lumbar segments. This finding suggests that in order to achieve clinically adequate treatment, spinal as well as supraspinal regions should be targeted for the cell engraftment.  This is further supported by a study by Xu et al., in which dual transplantation of human NSCs into the cervical (C4-C5) and lumbar (L4-L5) regions of the spinal cord prolonged survival of ALS rats by 17 days.  In a recent study, researchers at multiple institutions conducted 11 independent studies to test the efficacy of mouse and human NSC transplantation into ALS mice. Along with improvement in motor performance and a delay in disease progression, the study reported that 25% of the NSC-treated ALS mice survived for one year (three times longer than the untreated mice).  The reason for the variability in functional outcome of NSC therapy in different studies is unknown and may be related to the source and type of cell and the region of cell transplantation. Genetic modification of NSCs to enhance their migratory ability within the spinal cord or to increase the secretion of neurotrophic and anti-inflammatory biomolecules might further improve their efficacy. Studies showed that the transplantation of genetically modified NSCs overexpressing growth factors, such as GDNF or VEGF in ALS animals, significantly delayed disease onset, improved motor neuron numbers and functional outcome, and increased the survival time of the animals. ,
Two clinical trials are currently underway to test the safety and efficacy of intraspinal transplantation of NSCs in ALS patients. The first clinical trial was initiated in 2010 in the USA and phase I of the trial was designed to evaluate the safety and tolerability of the surgical procedure and the cell toxicity assessment is now completed.  In phase I, 18 patients with ALS received intraspinal NSC engraftment following a risk escalation paradigm. The patients were monitored for up to 2.5 years after the transplantation. Twelve patients (mean age 37-66 years, both non-ambulatory and ambulatory with forced vital capacity >60%) received either five unilateral or five bilateral microinjections into the lumbar spinal cord (L2-L5), and six patients received microinjection into the cervical spinal cord (C3-C5). No major adverse effects attributable to surgery or cells were noted. Although this phase of the trial was not designed to evaluate the efficacy of therapy, the authors reported no acceleration in disease progression and improved functional measures in one patient. , A Phase II open-label trial, which started recently, is currently enrolling participants by invitation and will assess the efficacy, feasibility, safety, toxicity, and maximum tolerated (safe) dose of the NSC therapy (ClinicalTrials.gov Identifier: NCT01730716). The second clinical trial, began in July 2012 in Italy, and is currently recruiting participants primarily to verify the safety of microsurgery and to evaluate the effect of therapy on the quality of life of the patients (ClinicalTrials.gov Identifier: NCT01640067).
Stem cell-based therapies hold great promise for ALS patients. Preclinical studies have highlighted the importance of different types of stem cells in ALS therapeutics. However, these findings also suggest that different cell types have different therapeutic effects (neuroprotection or cell replacement and regeneration), post-transplantation. Therefore, co-transplantation of different cell types in ALS may be an efficacious approach to achieve the maximum therapeutic effect. In addition, the success of any cell therapy depends on several critical issues, including route and accuracy of cell transplantation, long-term functionality of engrafted cells, and, most importantly, how cells interact with the host microenvironment. To address these questions, it is imperative to monitor transplanted cells. The current histological methods of analysis are highly invasive, require multiple tissue biopsies, and limit our ability to monitor transplanted cells in real- time over an elongated period of time. Several advanced non-invasive cellular imaging techniques to track engrafted cells in real-time are powerful tools for determining the efficacy of stem cell-based therapies  , and could be used in both ALS animals and patients. In clinics, although the use of stem cell therapy for ALS has gained considerable momentum in the recent past, there are still numerous hurdles that must be overcome. The course, severity, and clinical characteristics of the disease are extremely heterogeneous among ALS patients. Based on previous experience from unsuccessful pharmacological clinical trials, more stringent inclusion and exclusion criteria should be established. Optimal cell dose, route of delivery, and immunosuppressive regimen must also be carefully considered. It is equally important that these stem cell-based therapies must pass vigorous safety and quality control testing. To date, most countries do not have established standard protocols for cell expansion and storage, handling and shipping of cells, to check quality of cells at the time of administration, and evaluate long-term safety. Biologists, clinicians, government organizations, and industry experts should come together to introduce guidelines for more rigorous research practices, encourage more scientific clarity, and create stringent scientific standards that could guide the commercial development of stem cell-based therapies in the future.
[Figure 1], [Figure 2]