Motoneurons from human spinal muscular atrophy-induced pluripotent stem cells free of vector and transgenic sequences as a model and cell source for transplantation

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder and a leading genetic cause of infant mortality. Reprogramming adult human cells to induced pluripotent stem cells (iPSCs) allows generating patient-specific cells for disease modeling and therapeutic tools. However, this approach has required vector integration into the genome, limiting its applications. Here, we generated human iPSCs from a patient with a type 1 SMA and his father using non-viral, non-integrating episomal vectors. We obtained SMA and WT iPSC subclones free from exogenous sequences. The subclones showed morphologic and transcriptional similarities with ES (on microarray analysis) and differentiated into all three germ layers. IPSCs were differentiated into motoneurons that express specific motoneuronal markers. We found significant differences between SMA and WT motoneurons, including reductions in cell number, cell size, and axon length. We undertook a detailed assessment of transcriptional and splicing changes in motoneurons with microarray. We observed a relatively restricted number of genes, related to axon growth and motoneuron development, that presented a different splicing profile between SMA and WT cells. iPSC-purified motoneurons were transplanted into the spinal cords of SMA mice. We performed in vivo analysis evaluating whether and how iPSC-derived motoneurons integrated into the SMA spinal cord. We identified human-derived motoneurons within the ventral horns of all transplanted animals. Quantification data demonstrated that SMA motoneurons presented a reduced number of engrafted cells compared with WT. Motoneuron (WT and SMA) transplantation extends lifespan (> 50%) and ameliorates the phenotype of SMA mice. These results offer a proof of concept for the generation of patient-specific iPSCs and motor neurons free of exogenous elements.