Stem Cell Therapy in Pediatric Neurological Disabilities

Pediatric neurological disorders represent a major part of the disabilities worldwide. In over 10 decades of research to find a cure for these disorders, medical science has not been able to repair the underlying brain injury. This chapter focuses on recent advances in the application of stem cells as a therapeutic tool for some of the common neurode‐ velopmental disorders (cerebral palsy, autism, intellectual disability and muscular dys‐ trophy). The mechanism of action of stem cells in each disorder has been explained. A review of clinical data has been described giving a clear understanding of current status of stem cell therapy in these disorders. Various factors influencing the outcome of stem cell therapy such as different types of cells, different routes of administration and dosage and frequency of transplantation have also been discussed. Our experience of treating these disorders is exhibited in the form of our published data. Use of novel monitoring tools such as MRI MSK and PET‐CT scan brain to track the changes occurring at cellular level after stem cell therapy are described. We also highlight the importance of a multi‐ disciplinary approach of combining rehabilitation with stem cell therapy.


Introduction
Neurodevelopmental disorders (NDD) are characterized by an abnormal development of the brain during the early development phase, leading to a myriad of symptoms and diseases, including delayed milestones and deficits in personal and social functioning [1]. The developmental deficits can vary from specific limitations of adaptive, behavioral and cognitive functioning, motor dysfunction, to global impairments of social skills [2]. Some of the common neurodevelopmental disorders are cerebral palsy (CP), autism spectrum disorders, attention deficit hyperactivity disorder, intellectual disability (ID) or intellectual and developmental disability (IDD), learning disabilities, muscular dystrophies, Down's syndrome, genetic disorders such as fragile-X syndrome, spinal muscular atrophy (SMA) and metabolic disorders.
Pediatric neurological disorders represent a major part of the disabilities worldwide. In over 10 decades of research to find a cure for these disorders, medical science has not been able to repair the underlying brain injury [3]. The causes of NDD can be classified as congenital (present at birth) or acquired (developed after birth). The various etiologies are genetic defects, metabolic disorders, nutritional deficiencies, exposure to toxins, infections, hypoxia/ asphyxia, low birth weight, perinatal complications leading to traumatic brain injury or spinal cord injury in children [4]. This may affect language and speech, motor skills, behavior, memory, learning or other neurological functions affecting activities of daily life. While the severity of symptoms often change or evolve as the child's age progresses, these disabilities remain permanent. As these are lifelong disabilities, they pose a substantial economic burden on the society [5]. Hence, finding a treatment for them is the need of the hour. Improvement in the performance of these children would be of great significance to the quality of life of patients and their families.

Unmet medical needs
Therapeutic strategies and clinical expectations of patients and medical professionals have not yet been met. Currently, available treatments such as physiotherapy, occupational therapy, behavioral therapy, psychological intervention, speech therapy and pharmacological intervention only focus on alleviating the symptoms of these disabilities and do not address the underlying neuropathophysiology. However, the advent of stem cell therapy has opened new avenues for treatment of pediatric neurological disorders. In recent years, extensive research has been done to explore the potential of stem cells for the treatment of pediatric neurological disabilities. Until now, it was believed that once injured, the cells of the central nervous system cannot regenerate. However, owing to the distinct properties of stem cells to repair and regenerate, they can be considered as a potential therapeutic strategy.
This chapter focuses on recent advances in the application of stem cells as a therapeutic tool for some of the common NDDs (cerebral palsy, autism, intellectual disability and muscular dystrophy). The mechanism of action of stem cells in each disorder has been explained. A review of clinical data has been described giving a clear understanding of current status of stem cell therapy in these disorders. Various factors influencing the outcome of stem cell therapy such as different types of cells, different routes of administration and dosage and frequency of transplantation have also been discussed. Our experience of treating these disorders is exhibited in the form of our published data. Use of novel monitoring tools such as MRI MSK and PET-CT scan brain to track the changes occurring at cellular level after stem cell therapy is described. We also highlight the importance of a multidisciplinary approach of combining rehabilitation with stem cell therapy. Adverse effects of stem cell therapy are also enumerated.

What are stem cells?
Stem cells are blank, immature cells which have a capacity to self-renew and differentiate into host-specific multiple lineage cells [6]. Several types of stem cells are being explored for the treatment of neurological disorders such as bone marrow stem cells, embryonic stem cells, olfactory ensheathing cells and umbilical cord blood cells. The main aim of stem cell therapy is replacement of injured/dead neuronal cells and recovery of lost functions [7]. These cells perform repair process directly by regeneration of new cells or indirectly through paracrine activity. The chief underlying mechanisms of stem cells include neuroregeneration, neuroreplacement, neuroprotection, immunomodulation, axon sprouting and neural circuit reconstruction [8] (Figure 1).

Mechanism of action of stem cells in pediatric neurological disorders
Pediatric neurological disorders are caused due to mechanisms affecting the molecular, cellular and tissue plasticity of the brain and nervous system [9].
Stem cells when transplanted migrate and home towards the injured areas of the brain [10]. This homing property is attributed to the expression of growth factors, chemokine and extracellular matrix receptors on the surface of cells such as stromal cell-derived factor 1 (SDF-1), monocyte chemo attractant protein-3 (MCP-3), stem cell factor (SCF) and/or IL-8. They differentiate into the host tissue cells and replace the injured/dead neuronal tissue [11]. Through paracrine mechanisms they halt further injury and stimulate endogenous cells to carry out the repair and restoration process [12]. Stem cells secrete a vast array of neuroprotective growth factors including brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), glial cell line-derived neurotrophic factor (GDNF) and insulin-like  growth factor type 1. These growth factors activate a number of signaling pathways and help in enhancing differentiation, survival of neurons and maintaining neuronal functions [13]. They also produce vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (FGF-2) which improve perfusion and enhance angiogenesis [14]. Anti-inflammatory paracrine factors such as Interleukin 10 (IL 10) and Transforming growth factor (TGF)-β help in immunomodulation [15].

Clinical application of stem cell therapy in NDD
In this section, we discuss the literature review of various stem cell therapy studies in each disorder followed by our experience.
We published a study of 71 children diagnosed with different incurable neurological disorders. Autologous bone marrow-derived mononuclear cells were transplanted intrathecally and intramuscularly. Improvements were noted in muscle power, functional independent measure (FIM) and Brooke and Vignos scale. Imaging and electrophysiological investigations also showed improvement. Overall 97% muscular dystrophy cases showed subjective, functional and investigational improvement. Eighty-five percent of cases of cerebral palsy cases showed improvements. Eighty-eight percent of cases of other incurable neurological disorders such as autism, Retts syndrome and giant axonal neuropathy also showed improvement.
No major adverse events were noted.

Stem cell therapy in cerebral palsy
In cerebral palsy, white matter injury also known as periventricular leukomalacia (PVL) is one of the major pathologies observed [16]. Stem cells differentiate into neurons, oligodendrocytes and astrocytes which replace and repair the white matter injury in CP [17] (Figure 2). The growth factors secreted by these cells also help in remyelination, synaptogenesis, cytoprotection and angiogenesis which reverse the cellular injury in CP [18,19]. Numerous preclinical studies have Physical Disabilities -Therapeutic Implications demonstrated the potential of stem cell transplantation in cerebral palsy. The homing property of these cells was confirmed by Chen et al., who transplanted magnetically labeled mesenchymal stem cells in a model of perinatal brain injury and found that these cells migrate to lesion sites and proliferate [20]. Studies have demonstrated the differentiation of bone marrow, umbilical cord blood, neural and other progenitor stem cells into neurons and oligodendrocytes in experimental animal models [21][22][23][24][25]. Transplantation of stem cells in rat models have resulted in improved cognition and sensorimotor deficits along with functional recovery [26].

Clinical evidence
In cerebral palsy, around 26 studies have been published explaining the effect of stem cell therapy. Overall, 579 (90%) out of 646 patients have shown improvements ( In 2015, we published a nonrandomized study demonstrating the benefits of autologous bone marrow mononuclear cells (BMMNCs) in cerebral palsy [27]. These patients were followed up at 3 and 6 months. Six months after intervention, 38 out of 40 (95%) patients showed improvements and 2 did not show any improvement but remained stable without any deterioration ( Figure 3). No major adverse events were noted except for seizures in two patients which were controlled by medications.

Physical Disabilities -Therapeutic Implications
We have also published three case reports demonstrating the safety and efficacy of BMMNC transplantation in cerebral palsy [36,37,39]. In these case reports, the functional improvements are supported by improved brain metabolism recorded in comparative PET-CT scans performed before and after the intervention.

Stem cell therapy in autism
In autism, immune dysfunction, hypoperfusion, oxidative stress, decreased number of Purkinje cells (PCs), cerebellum alterations, defective cortical organization and altered plasticity of dendritic spine morphology are the underlying neuropathologies (Figure 4) [52,53]. Stem cells modulate the immune dysfunction by releasing anti-inflammatory molecules and inhibiting pro-inflammatory molecules, which further reduces neural injury [54]. They also facilitate angiogenesis which increases blood and oxygen supply to the brain thus reversing the hypoperfusion [55]. Stem cells may also reinforce cortical plasticity, promote synaptic plasticity and restore cerebellar PCs [56]. These mechanisms collectively may improve the lost neural connectivity and restore lost functions in autism. In an experimental model of mice, H Segal Gavish et al. transplanted mesenchymal stem cells, which resulted in reduction of stereotypical behaviors, decrease in cognitive rigidity and improvement in social behavior. Tissue analysis revealed elevated BDNF protein levels in the hippocampus accompanied by increased hippocampal neurogenesis in the MSC-transplanted mice compared with sham treated mice [57].

Clinical evidence
A total of 11 studies (3 case series and 8 case reports) have been published all over the world demonstrating the benefits of stem cell therapy in autism. Overall, 122 patients were administered with cellular therapy and 90 showed improvements ( Table 2) [58][59][60][61][62][63][64][65][66][67][68].   Physical Disabilities -Therapeutic Implications scores and 20 patients (62%) showed decreased severity on CGI-I. On CGI-II 96% of patients showed global improvement. Improvements in brain metabolism were also observed on positron emission tomography-computed tomography (PET-CT) scan brain. All 32 patients were monitored through the duration of follow-up for any major adverse events. Incidence of seizures was recorded in three patients, which were reversible and easily controlled with medications.
In addition to the above study, we have also published eight case reports demonstrating the safety, efficacy and objective improvements on PET-CT scan brain in patients with autism following stem cell therapy [61][62][63][64][65][66][67][68].

Stem cell therapy in intellectual disability
In intellectual disability (ID), the neuronal connectivity in the brain is impaired along with disrupted cell migration, cell multiplication, axon growth, brain plasticity and synaptogenesis ( Figure 6) [9]. Studies have recorded defects in hippocampus and cerebral cortex areas of the brain leading to faulty information processing, consecutively affecting cognition and adaptive behavior in ID. Stem cells restore the synaptic transmitters released and provide local reinnervations to the area affected. It also integrates existing neural and synaptic network and re-establishes connections of functional afferent and efferent cells which may have contributed in restoring the cognitive and functional deficit in IDs [69].

Clinical evidence
We are currently under process of analyzing the data of a prospective study conducted to demonstrate the effect of autologous bone marrow mononuclear cells in intellectual disability.  However, in 2015, we published a report of a 13-year-old boy with intellectual disability who exhibited improvements after stem cell therapy [70]. He was followed up after 3 and 6 months of intervention. No major adverse events were recorded post intervention. Over a period of 6 months, he showed improved eye contact, cognition, learning ability, behavior and ability to perform activities of daily living. His score on Functional Independence Measure (FIM) increased from 67 to 76. On comparing the pre and post PET-CT scan, improvement in metabolic activity of hippocampus, left amygdala and cerebellum was recorded. These changes correlated to the functional outcome.

Stem cell therapy in Duchenne muscular dystrophy
The underlying pathogenic mechanism of muscular dystrophy is an imbalance between muscle degeneration and resident satellite cell-mediated regeneration [71]. Satellite cells, the adult skeletal muscle progenitor cells, are considered to be the main cell type involved in skeletal muscle regeneration. Continuous cycles of degeneration and regeneration of muscle fibers exhausts the muscle stem cell pool, leading to muscle being replaced by adipose and fibrotic tissue. Stem cell therapy holds great promise as a treatment for Duchenne muscular dystrophy by providing cells that can both deliver functional muscle proteins and replenish the stem cell pool [72].
Stem cells are known to enhance angiogenesis, contribute to neovascularization, promote tissue remodeling, prevent apoptosis, decrease inflammation, release growth factors and activate the satellite cells [73] (Figure 7). In animal models, these cells have shown to produce the deficient proteins and make new muscle cells which fuse with the host fibers. Further, stem cell-derived exosomes which are small membrane vesicles and are responsible for inter-cellular communication, promote muscle regeneration by enhancing myogenesis and angiogenesis [74].

Clinical evidence
A total of 14 studies have been conducted demonstrating the efficacy of stem cells in muscular dystrophy. Various types of stem cells such as bone marrow-derived cells, umbilical cord stem cells and muscle-derived cells were used. Out of a total of 346 patients who underwent stem cell therapy, 296 showed a positive outcome ( We conducted a study on 150 patients diagnosed with muscular dystrophy. On a mean follow up period of 12 months ± 1 month, 86.67% cases showed symptomatic and functional improvements, with six patients showing muscle regeneration and decrease in fatty infiltration on musculoskeletal magnetic resonance imaging (MRI MSK) and nine showing improved muscle electrical activity on electromyography (EMG). Fifty-three percent cases showed increase in trunk muscle strength, 48% an increase in upper limb (UL) strength, 59% an increase in lower limb (LL) strength and about 10% showed an improved gait pattern (Figures 8 and 9).

Adverse events of stem cell therapy
The adverse events following stem cell transplantation mainly depends on the type of stem cell and the route of administration. Other factors like dosage of cells, frequency of transplantation and age of the patient may also contribute. Fetal stem cells are known to be potentially tumorigenic [91]. Use of umbilical cord stem cells is limited due to slow or incomplete immune reconstitution, resulting in a high transplantation-related mortality (TRM) due to infections. Most studies have demonstrated a predominance of Gram-positive bacteria (GPB) bloodstream infections [92]. On the contrary, adult stem cell has not shown any serious adverse events. Autologous cell transplantation is safer than allogenic.
Adverse events of stem cell therapy can be categorized into minor and major adverse events. Minor adverse events include procedure related events such as spinal headache, nausea, diarrhea, vomiting, pain or bleeding at the site of aspiration/injection and fever amongst others. These are treated using medications. Anesthetic complications and allergic reactions may also occur depending on the procedure. Major adverse events include episodes of seizures occurring after intervention. These can be managed prophylactically. Pre-existing epileptogenic focus in Electroencephalogram (EEG) also predicts the occurrence of seizures. Evidence suggests that antiepileptic prophylactic regimen decreases the incidence of seizures as an adverse event after stem cell therapy [93].

Routes of administration
The route of delivery of cells plays an important role in maximizing the clinical output of cellular therapy. Intrathecal route of administration is a relatively minimally invasive and targeted route of administration of cells. It is devoid of any major side effects [94]. In neurological disorders, intrathecal transplantation enhances the accessibility of the injected cells into the CNS [95]. Intramuscular injections are administered at the motor points plotted on the affected muscles. Motor points are the points where the innervating nerve enters the muscle. Thus, implantation of cells in the muscles enhances the effect of stem cells on the degenerating muscles [96]. Intravenous administration is the least invasive route. However, evidence suggests that majority of cells get trapped in the pulmonary passage and only few cells reach the injured site [97]. An alternate route of administration is via intra-cerebral route. But, it is an invasive technique and might result in secondary complications such as bleeding and neural tissue injury [98]. Hence, as compared to all the delivery routes, intrathecal administration is most efficacious.

Types of cells
Cells used from allogenic sources have an inherent risk of immunogenicity and may potentially cause immune rejection of graft versus host disease. Autologous cells have the least possibility of immune reaction and so far clinical studies with autologous minimally manipulated cells have shown no immunogenic reactions in the host post transplantation. Autologous cells may therefore be a safer option in children with NDD.

Etiology
Genetic factors play a major part in the pathology of neurological disorders and gene therapy has provided novel insights in treating the underlying genetic aberrations. But gene therapy cannot replace the lost neurons and practical difficulties have prevented it from being a clinically feasible and viable option at present. The sporadic nature of the disease is also an important factor influencing the outcome, where the etiology of the disease is unknown. Stem cell therapy addresses the core injury occurring in the brain. The multiple mechanism of action of the stem cells addresses the multifactorial pathology of the NDD.

Severity
It has been observed that the mild cases of neurodevelopment disorders have a better recovery curve than the chronic cases. In mild cases, axonal function remains intact and recovery can be rapid if remyelination occurs. In severe cases, axonal degeneration occurs and recovery depends on axonal regeneration. Recovery becomes much slower, and there is a greater degree of residual injury. Mild cases require lesser dosage of cells and the frequency of doses required is less to attain potential recovery than the severe cases.

Age of the patient
One of postulated hypothesis is that the neural circuits, that form the basis for learning, behavior and health, are more plastic during the initial years of life. They become increasingly difficult to alter over time. Age-related decline in the potency of the stem cells is observed which might also affect the remodeling of CNS by these cells. Early intervention is advised for better outcome of stem cell therapy.

Importance of neurorehabilitation
Neurorehabilitation aims at restoration and maximization of functions that have been lost due to impairments caused by injury or disease of nervous system making the patient functionally independent. The rehabilitation regime promotes and facilitates neural plasticity [99]. Studies have shown that exercise enhances the effect of injected stem cells by inducing mobility of the cells, activating and proliferating the local stem cells, promoting muscle angiogenesis and release of cytokines and nerve growth factors. Hence, neurorehabilitation compliments with the stem cell therapy [100].

Objective evidence: neuroimaging techniques to monitor the outcome of stem cell therapy
Neuroimaging techniques enable the quantitative measurement of various biological markers which may serve as a powerful tool for optimizing the use of stem cells for clinical applications.

Positron emission tomography (PET-CT) scan brain
PET-CT scan brain can be used efficiently as a monitoring tool to study the outcome of stem cell therapy. One of the advantages of using PET-CT is its extreme sensitivity enabling it to detect molecules at the nanomolar level [101]. Brain 18F-FDG PET allows studying the cerebral glucose metabolism, indicating the neuronal and synaptic activity. It dynamically measures the energy metabolism along with blood oxygenation and blood flow [102]. The alteration in neuronal activity caused by disease is reflected in change of glucose metabolism and can be revealed in the PET-CT scan brain. As mentioned previously in the clinical results, there were improvements recorded in the brain metabolism of patients included in the clinical studies. The changes seen on PET-CT scan brain correlated with the clinical improvement indicating that it can identify alteration occurring at the tissue levels (Figures 10-12).

Magnetic resonance imaging (MRI)
Magnetic resonance imaging (MRI) is gaining popularity because of its capacity to reveal characteristic findings that address the diagnosis and support therapeutic interventions.  Since MRI is devoid of ionizing radiations, it has turned out to be a valuable imaging method in children, although sometimes sedation might be necessary. In the past few years, studies have reported on the detection of muscle involvement pattern in various muscular dystrophies through MRI musculoskeletal imaging (MRI/MSK). The images provide a high soft tissue contrast allowing assessment of affected striated muscles in terms of shape, volume (hypotrophy and hypertrophy) and architecture [103,104]. MRI MSK was used as a tool to  assess the therapeutic efficacy of stem cell therapy in muscular dystrophy. In our published data, images of MRI MSK performed after intervention has revealed stabilization of disease progression in muscular dystrophy.

Conclusion
In children, the brain is still at a developing stage and not fully matured resulting in maximal neural plasticity during childhood. Hence, likelihood of improvement in affected areas of the brain increases manifold with early intervention. Stem cell therapy has recently gained lot of importance as a therapeutic strategy for various disorders including NDDs.
In this review, we have demonstrated the outcome of stem cell therapy in NDDs mainly cerebral palsy, autism, intellectual disability and muscular dystrophy supported by our published data. Through its neurorestorative and neuroregenerative property, stem cells have the capacity of repairing the underlying neural and muscular dysfunction. This property can augment neurodevelopment, facilitating achievement of milestones earlier as compared to the current conventional treatment modalities. In progressive developmental disorders like muscular dystrophies, stem cell therapy has shown to slow down the disease progression. The data also establishes the fact that autologous stem cell therapy is a safe and efficacious treatment which helps in recovery of lost functions and neural plasticity.
Though stem cell therapy is not a cure, the gap between normalcy and disability can be minimized. Stem cells in combination with the multidisciplinary medical and rehabilitative modalities can enhance and hasten the recovery from NDD which will help the patient to lead a productive and respectable life in the society.

Future directions
Stem cell therapy is still in its developing stage. There are still numerous uncertainties prevailing with respect to optimum volume of cells to be injected, number of doses, route of administration, types of cells amongst others. The advent of induced pluripotent stem cells (iPSCs) has provided opportunities for the study of human neurodevelopmental diseases in a controlled environment. Reprogramming cells from patients with neurological diseases will allow the study of disease-specific cellular and molecular pathways causing these diseases. Also, the establishment of neural stem cells (NSCs), a life-long source of neurons and glia, has contradicted the dogma that the nervous system lacked regenerative power. Future studies need to focus on the precautionary pre-intervention assessments to identify patients with high risk for seizures and related adverse events after stem cell therapy. A better knowledge of all these factors will improve the therapeutic effectiveness of stem cell therapy. Future studies should consider the use of modern radiological tools as monitoring