Transcriptomic and functional insights across different genetic mutations of ALS iPSC - derived neural progenitors and motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal and heterogeneous neurodegenerative disorder affecting upper and lower motor neurons (MNs). Its clinical variability and unclear molecular mechanisms continue to hinder therapeutic development. There is currently no cure; existing drugs aim to slow disease progression but show limited efficacy and tolerability, highlighting the need for alternative strategies. Stem cell-based approaches are emerging, but results remain modest. Induced pluripotent stem cells (iPSCs) are widely used in preclinical ALS research. In this study, iPSCs were generated by reprogramming CD34+ cells from the peripheral blood of healthy donors and ALS patients carrying different mutations (C9orf72, TARDBP, KIF5A). These iPSCs expressed typical pluripotency markers (OCT4, SOX2, KLF4, c-MYC, SSEA3, TRA1-60) and were able to differentiate into the three germ layers. Transcriptomic analysis of iPSCs and derived neurospheres revealed a downregulation of extracellular matrix (ECM) genes in ALS-iPSCs, suggesting a possible early shared pathogenic signature. Neurospheres showed broader transcriptional impairments in neuronal development pathways such as axonogenesis, synaptic membrane formation, and neuron projection development. However, these changes varied across patients, reflecting genetic heterogeneity. Functionally, ALS neurospheres displayed reduced neurite branching and network complexity. Differentiation into MNs confirmed a reduction in neuronal density, axonal length, and diameter in ALS-derived MNs. Furthermore, ALS-MNs exhibited lower levels of key maturation markers ( as Chat, TUBB3, MAP2, SYP, SYT, NeuroD1, NFH, NaV1.1, Kv2.1), indicating impaired neuronal maturation. This work contributes to understanding ALS pathogenesis and supports the relevance of iPSC-based models in developing future therapies.