Cellular Therapy for Spinal Muscular Atrophy: Pearls and Pitfalls

Introduction

Spinal muscular atrophies (SMAs) include a series of neuromuscular genetic diseases in which spinal motor neurons degenerate leading to progressive paralysis with proximal muscular atrophy. The most frequent condition is caused by mutations of the survival motor neuron 1 ( SMN1 ) gene on chromosome 5q13 . The human genome also contains a highly homologous gene on the same chromosome, called SMN2. It differs from SMN1 by the presence of a C→T nucleotidic replacement in a critical position on exon 7. This results in an altered splicing of the transcript leading to the production of a very small amount of SMN full-length functional protein.

The 5q form of SMA represents the most common genetic cause of death during childhood affecting about 1 in 10,000 newborns. SMA clinical manifestations include muscular weakness and atrophy especially affecting lower limbs and proximal segments. Muscular wasting leads to severe difficulties in breathing and swallowing; death usually occurs due to cardiorespiratory failure. The number of copies of the paralogous gene, SMN2 , which can contribute only a small amount of SMN full-length protein, appears to determine the severity of the clinical picture. The SMA type 1 form represents the most frequent and severe phenotype; affected children manifest the disorder within 5–6 months after birth, and never reach motor-developmental milestones. They cannot sit or walk autonomously, and require early ventilator support. SMA type 2 displays a milder clinical phenotype; patients become symptomatic between 6 and 18 months from birth and are able to sit independently. SMAs 3 and 4 become overt later (the latter during adulthood), and while patients display symptoms related to muscular weakness and altered motor performance, their life expectancy is comparable to the general population. There is no effective treatment clinically available for SMA patients, who require a 24-h assistance with specific sanitary devices. A multidisciplinary environment in which clinicians, physiotherapists, psychologists, and speech therapists work collectively currently represents the most proper supportive approach. Due to its high frequency, monogenic etiology, and severe clinical and social burden, SMA represents a valuable target for experimental therapeutic approaches.

Potential Therapeutic Effects of Stem Cells on Spinal Muscular Atrophy Disease Mechanisms

Although the genetic origin of SMA has been revealed during the last decade, many questions still remain about downstream SMN1 mutation molecular pathogenic mechanisms. The precise physiological role of SMN protein is the focus of several ongoing researches. SMN appears to complex with Gemin proteins forming a structure that cooperates into the biogenesis of uridine-rich small nuclear ribonuclear proteins. They are a crucial contributor to spliceosome formation and splicing process. Other functions have been attributed to SMN including a role in axon maturation and myelination. The rather selective vulnerability of motor neurons to SMN reduction remains a major issue to be addressed.

Several research groups have exploited the murine model to investigate pathological mechanisms involved in SMA development. One model has been obtained by introducing the human SMN2 transgene on a Smn-null background. The so-called SMNΔ7 mice mimic SMA1 human phenotype in terms of neuromuscular waste, with a rather selective involvement of spinal motor neurons. However, SMNΔ7 animals display a systemic multiorgan involvement with distal tissue necrosis, which is more severe than it appears to be in humans, and it significantly affects mouse life span. This and other differences are rather species-specific and need to be taken into account during the analyses of the experimental data. The discovery of human pluripotent stem cells and their use in disease-modeling-studies has led to the precious possibility of investigating pathogenic mechanisms peculiar to human pathology.

Several studies on murine models and human pluripotent stem cells have highlighted widespread axonal and synaptic alterations in SMA motor neurons, including scarcity of dendrites and spines, impairment of calcium metabolism and dysfunctional remodeling ability. SMA human pluripotent stem cells are able to differentiate into motor neurons but derived cells are characterized by a reduced life span; a crucial role of apoptosis is noted in the disease onset, with increased caspase-8 and-3 activation.

Moreover, other cell types beside motor neurons have been recently implied in SMA onset. Interneurons and sensory neurons have been shown to be affected in the disease course thus suggesting the SMN loss could lead to a widespread perturbation of the neural network.

Non-cell-autonomous mechanisms due to a toxic activation of glia cells are emerging as a central contributor in motor neuron death. Astrogliosis has been known to play a pathogenetic role in amyotrophic lateral sclerosis (ALS), and recent discoveries have pointed out that SMA astrocytes could trigger motor neuron degeneration, both by losing their trophic function and acquiring inflammatory features. Overall, SMA pathogenesis appears to involve complex molecular mechanisms in multiple cell types highly interactive in creating a pathological microenvironment.

In this context, stem-cell-based therapeutic approaches could counteract several disease mechanisms at the same time. Pluripotent stem cells are able to give rise to mature motor neurons reacting to exogenous signals and thus replacing lost cells after engraftment, similar to what happens during physiological neurogenesis after injury. Stem cells are also able to differentiate in glia lineage and substitute toxic astrocytes with healthy ones, which are able to provide neuroprotection to endogenous motor neurons. Indeed, transplanting neural stem cells (NSCs) could be more effective than transplanting more mature cell subtypes. NSCs are more robust and intrinsically plastic; they can give rise to different subpopulations of healthy cells to replace and sustain endogenous ones. They could contribute to alleviate the neural network impairment by building alternative circuitry and stimulating the formation of new synapses. Furthermore, the role of oligodendrocytes and myelination dysfunction in SMA pathogenesis as potential therapeutic targets has to be further investigated. As a consequence, stem cell-based therapeutic approaches could be clinically effective through a multifactorial action consisting of modulation of glial impairment, trophic sustainment of the endogenous cells, and enrichment of the unhealthy microenvironment.

Indeed, several methods exploit stem cells in order to give rise to de novo production and exogenous import of neurotrophic molecules to the diseased spinal cord. Human NSCs are able to express several trophic factors and can be manipulated to produce specific substances. Insulin-like growth factor-I (IGF-I), brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) have been demonstrated to alleviate neurodegeneration and provide environmental support in a series of models of neurodegenerative diseases. Moreover, the SMA nervous system showed an impairment in IGF1 signaling, which could represent a valid target for this approach in stem cell-mediated therapy.

Overall, these considerations support the hypothesis that cell-based therapies may function in supporting SMA motor neurons, especially by providing a trophic and protective environment within the spinal cord, and thus counteracting the multifactorial pathogenetic mechanisms of neuronal death. Stem cell transplants could represent a complementary approach to SMA molecular and gene therapies. The latter are able to precisely target the loss of SMN restoring its function, but they could have a minor impact on later-diagnosed patients, while cell therapy could be useful in managing symptomatic phases of the disease. However, it is still worth considering that mechanisms downstream of stem cell activity are poorly understood, as are the molecular pathways underlying neural repair. Elucidating these aspects will provide invaluable insight into SMA pathogenesis and possibilities for therapy, which could be suitable also for other motor neuron diseases.

The Selection of Cell Types to be Transplanted

During the last decades, a few relevant preclinical studies have been conducted on the use of different cell subtypes for SMA therapy. Cells have been screened for their ability to target the injured area, survive, and correctly engraft exerting a therapeutic action.

Many of these studies employed NSCs or derived motor neurons. A series of relevant preclinical studies have been reported (see Table 11.1 ). Hopefully, these results could open the way to clinical translation, even though more experiments will be necessary to accurately assess safety and effectiveness of stem cell based approaches.

Embryonic Stem Cell Derived Motor Neuron Precursors for Spinal Muscular Atrophy

A novel technique to obtain highly enriched human motor neuron precursor (hMNP) cultures was established by California Stem Cell Inc. HMNPs were derived from human embryonic stem cell (ESC) lines that were expanded on Matrigel for the first 3 weeks in a media supplemented with basic fibroblast growth factor (bFGF) . Cells were then transferred to ultralow binding dishes and suspended in MN differentiation media, including Glutamax, B27, insulin, sodium selenite, transferrin, MgSO4, and bFGF. Cells were exposed to the differentiation media for 5 days, supplemented with retinoic acid (RA), and then fully characterized. The transplantation of derived cells in the spinal cord of SMA models resulted in a proper engraftment and survival. Moreover, transplanted hMNP were able to produce trophic factors in situ (i.e., neurotrophin-3 (NT-3) and nerve growth factor). The number of endogenous motor neurons was significantly increased. Given these results, the research group designed a clinical trial, but until now, the FDA has not approved the study.