Study of cell therapy for ALS using neuronal stem cells from iPSCs with ectopic SOX2 expression

Abstract

Amyotrophic Lateral Sclerosis (ALS), a regressive neuronal disease, results in the death of motor neurons. It is known as a deadly disease and most ALS patients die within five years of diagnosis due to its rapid progression, however, there are few treatments available for ALS. In order to attempt to develop a cell therapy system for the treatment of ALS, here, we use neuronal stem cells (NSCs) overexpressing the sex determining region Y-box 2 (SOX2). NSCs are differentiated from induced pluripotent stem cells (iPSCs), in particular, SOX2 is related to the differentiation into a neuronal lineage. Therefore, we focus on the overexpression of SOX2 in relation to the potential of NSCs to differentiate into motor neurons, and their effect on ALS in a mouse model. NSCs contain an inducible system to overexpress SOX2, which can be controlled by the use of doxycycline, therefore we gauged the differentiation potential by the level of SOX2 expression. According to quantitative RT-PCR and immunocytochemistry, motor neurons were more differentiated with increasing levels of doxycycline. To evaluate the functional activity of differentiated NSCs, they were transplanted into the ALS Cu/Zn superoxide dismutase 1 (SOD1) G93A transgenic mouse model (SOD1 G93A TG). The NSCs were injected at the junction between the brain and spinal cord, engrafting and surviving successfully, and further treated with doxycycline. A greater differentiation into motor neurons was expected in the group of treated mice, however the NSCs were differentiated into various other subtypes of neurons and glial cells, including astrocytes, after migration from the injection site to other areas of the brain and spinal cord. For the functional analysis of transplanted NSCs, we tested the movement and life span of SOD1 G93A TG mice engrafted with NSCs. Compared with the untreated control mice, the motion of the mice transplanted with NSCs was not improved, and their life span was not significantly extended. However, the NSCs prolonged the disease onset point. As the number of remaining cells reduced until death, the effectiveness of the NSCs was not carried over to the end of disease progression. With further experiments, considering the number of injection times and cells, the lifespan and motor function will likely be improved. Therefore, the NSCs that overexpressed SOX2 were well differentiated into motor neurons, although the ALS mouse did show extensive neural differentiation of differing types. In addition, NSCs were effective in slowing the ALS disease onset.