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

BACKGROUND: Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Reprogramming adult human cells to induced pluripotent stem cells (iPSCs) makes it possible to obtain patient-specific cells for disease modeling and therapeutic tools. However, this approach has required vector integration into the genome, limiting its applications. OBJECTIVE: To describe the generation and application of human spinal muscular atrophy (SMA)-induced pluripotent stem cells (iPSCs) and motoneurons using non-integrating episomal vectors. DESIGN/METHODS: We generated iPSCs from fibroblasts from a patient with SMA and his unaffected father using a non-viral method. Cells were transfected with oriP/EBNA1 vectors encoding six reprogramming factors. We differentiated iPSCs using a multistage protocol to promote motoneuron commitment, based on the use of SHH, RA, and other morphogens. The phenotype of differentiated cells was studied by morphological, gene expression, and protein analysis. iPSC-purified motoneurons were transplanted into the spinal cords of SMA mice. Histochemical and neuropathological analyses were performed. Survival and neuromuscular function were investigated. RESULTS: We successfully isolated SMA and WT iPSC subclones free from vectors and exogenous sequences. Morphological, immunocytochemical and genome-wide expression analysis confirmed the reprogramming of the cells to a pluripotent state capable of generating the three germinal layers in vitro and in vivo. IPSCs were differentiated into post-mitotic motoneurons that express motoneuron-specific transcription factors such as HB9 and Isl1 as well as ChAT. We detected significant differences between SMA and WT motoneurons, including reductions in cell number, cell size, and axon length. Our next goal was to determine whether MNs derived from iPSCs survive and engraft appropriately within the SMA spinal cord. We identified human-derived motoneurons, which presented motoneuronal phenotype and coexpressed HB9 and ChAT, within the ventral horns of all transplanted animals. Quantification analysis demonstrated that SMA motoneurons presented a reduced number of engrafted cells compared with WT. Transplantation of wild-type and SMA motor neurons extended the lifespan and ameliorated the phenotype of SMA mice. CONCLUSIONS: These results offer a proof of concept for the generation of patient-specific iPSCs and motor neurons free of exogenous elements with potential value for research and clinical applications