Neural Stem Cells as a Therapeutic Approach for Amyotrophic Lateral Sclerosis

Proliferating neural stem cells (NSCs) were first identified in the late 1960s in the adult rat brain¹ as multipotent self-renewing stem cells, able to differentiate into neurons, astrocytes, and oligodendrocytes. NSC transplantation is being evaluated to treat traumatic brain or spinal cord injury and neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is a fatal neuro­degenerative disorder characterized by progressive loss of upper and lower motor neurons and chronic inflammation leading to paralysis and death due to respiratory failure. In the December issue of Science Translational Medicine, Teng et al.² reported successful use of transplanted, undifferen­tiated multipotent NSCs to prolong survival in a mouse model of ALS. They found that the experimental treatment is both safe and potentially beneficial to preserve neurons that have been spared by the disease at the time of treatment. The results further indicate the benefits of early intervention and suggest that targeting the spinal cord at different sites will protect both motor and respiratory function. The authors conclude that a combination of therapeutic approaches, from gene therapy to cell transplantation, targeting different pathogenic mechanisms of the disorder, is likely to lead to successful therapy.

ALS is largely sporadic in origin, with only about 10% of total cases having a family history of the disease. The genetic causes are known for about 50% of familial cases; the only risk factor clearly associated with the disease is aging.³ Mutations in the superoxide dismutase 1 (SOD1) gene are one of the most common causes of familial ALS, and transgenic animals carrying the mutant gene recapitulate the pathophysiology of ALS. Although the main feature of the disease is motor-neuron death, in recent years it has become clear that the nonneuronal cells surrounding the vulnerable neurons play a crucial role in disease progression and represent a promising therapeutic target.⁴ However, drug therapies aiming at targeting single cell types and/or single pathogenic mechanisms have failed to provide a successful therapeutic outcome. The development of a cell-based therapy able to target multiple components of the affected system could be beneficial by both supporting dying neurons and improving the milieu of the spinal cord by secreting neurotrophic and anti-inflammatory factors. Early studies in rodent models of ALS have shown that transplantation of human NSCs can transiently improve motor performance as well as moderately increase survival without major side effects.^5,6

Teng et al. made use of a highly standardized protocol and common experimental material at four institutions, involving 11 independent, double-blinded studies, to evaluate the therapeutic effects of mouse and human NSC transplantation into the mutant SOD1 mouse model. In a first series of experiments, investigators transplanted nestin-positive, multipotent mouse NSCs into one site in the lumbar spinal cord of two groups of ALS mice: early-to-mid-symptomatic stages and late end stage. The single injection resulted in robust and extensive integration of donor cells in the lumbar cord, 99% of which were non­neuronal cells. Additionally, coordinated hind-limb function evaluated by the rotarod test was improved in the early-to-mid-symptomatic group and survival was 10 days longer than in the noninjected controls. The authors determined the mechanisms by which transplanted mouse and human NSCs were acting to delay host-neuron degeneration by observing that NSCs secreted increased levels of the trophic factors nerve-growth factor, brain-derived neurotrophic factor, and glial cell line–derived growth factor (GDNF), all of which enhance motor-neuron survival.

Next, the authors sought to evaluate the therapeutic effects of human NSCs (hNSCs) and the effects of intervening at earlier time points. The injections of hNSCs were performed bilaterally at one site (L₂) or multiple sites (C₆, T₁₀, L₁, and L₃) in the spinal cord of mutant SOD1 mice at 8–10 weeks of age. Although clinical signs are not yet evident at this age, histopathology reveals clear signs of neurodegeneration. After NSC injection at a single site (L₂), motor performance improved and there was a mild increase in survival; the multisite approach showed more encouraging results. The authors report a remarkable survival increase of 200 days or up to a full year in 40% and 20%, respectively, of animals injected with hNSCs at all four sites of the spinal cord during the presymptomatic stage of disease. Interestingly, the remaining 40% of mice injected with hNSCs showed a very mild increase in survival. These findings may be caused by variation in the level of hNSC engraftment achieved in segments of the spinal cord regulating vital functions such as respiration. Despite the survival variability, meta-analysis of these studies produced an estimated hazard ratio of 0.33, meaning that the risk of death of treated animals was 67% lower than in controls.

Interestingly, this large study confirmed that the beneficial effects of this therapeutic approach derived mainly from the ability of undifferentiated NSCs to secrete growth factors that protect spared motor neurons. Further studies are needed to identify the factors associated with transplants that result in significant increases in survival. It will also be useful to assess survival of the transplanted cells over time and to determine whether multiple injections at different stages could further improve survival or if the transplanted cells are negatively affected by the local disease environment. Recent studies suggest that ALS might originate focally and then spread through the spinal cord in both sporadic and familial cases.⁷ Moreover, this evidence has led to the hypothesis that misfolded SOD1 might be acting as a prion-like protein, propagating the disease from cell to cell.⁸ In this case, the beneficial effects of wild-type transplanted cells would be transient, and they could also acquire toxic properties. Interestingly, Teng et al. found no improvement when NSCs were injected in late-symptomatic animals. As the authors suggest, this result could be due to the already extensive loss of motor neurons, and it would be interesting to investigate whether the cells implanted at later time points present a secretome and functional characteristics that are similar to that of the NSCs implanted at earlier stages or whether the more aggressive environment of the spinal cord at later stages of the disease inhibits survival and function of the transplants.

As Teng and colleagues suggest, NSCs have the potential to be a valuable therapeutic vehicle because they are site-specific and become integrated into the parenchyma. However, engineering the NSCs to increase their migratory power within the spinal cord or to enhance their secretion of neurotrophic and anti-inflammatory factors might increase their efficacy. Human NSCs and mesenchymal stem cells engineered to secrete high levels of GDNF have already been shown to have beneficial effects in animal models of ALS.⁹ However, unilateral injection of engineered hNSCs in the spinal cord of ALS rats protected dying motor neurons but not their projection to muscle, with minimal impact on motor performance and survival.¹⁰ By contrast, injection of mesenchymal stem cells secreting high levels of GDNF in the muscles of ALS rats protected the neuromuscular junction and significantly increased survival.¹¹ This suggests that NSC injections in the spinal cord might lead to a better outcome if combined with other therapies aimed at preserving the neuro­muscular junction.

Two clinical trials are currently testing the safety and the effects of intra–spinal cord delivery of hNSCs in ALS patients. The first was initiated in 2010, and phase I is now complete.¹² Twelve patients received either 5 unilateral or 5 bilateral (10 total) injections into the lumbar spinal cord at doses of 100,000 cells per injection. This first trial was designed with the aim of evaluating safety and tolerability of the procedure in ALS patients, and thus far there is no indication that the cells are toxic or injurious to the spinal cord. Although this phase of the trial was not aimed at evaluating efficacy, the authors report mild but encouraging improvements in terms of disease progression. However, as suggested by the study by Teng et al.,² the aim of the next phase of this clinical trial will be targeting the cervical spinal cord to preserve respiratory function and increase patient survival. The other trial, which began in July 2012, is currently recruiting participants with the goal of evaluating safety as well as clinical outcomes (http://clinicaltrials.gov/ct2/show/NCT01640067?term=neural+stem+cells+and+als&rank=1).

In summary, stem cell transplantation for severe diseases such as ALS shows preclinical signs of modest improvements. The field continues to make advances in delivery of cells to the spinal cord as well as in understanding the mechanisms for the beneficial effects seen in the rodent models, highlighting the importance of continued research and development in this area.