Abstract
Induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) offer a promising model system for understanding motor neuron diseases (MNDs) and advancing drug discovery. However, variability in differentiation outcomes presents a major barrier to reproducibility and model reliability. This study evaluates a widely adopted small molecule protocol for MN differentiation to quantify variability and identify its sources within an industrial setting. Analysing data from 15 differentiation sets across 8 cell lines, we found that non-genetic factors - particularly induction set and operator - were the predominant sources of variability, outweighing the contribution from cell line genetics. We further demonstrated that iPSC genomic instability, as assessed by a targeted RT-qPCR assay for common karyotypic abnormalities, significantly affected differentiation efficiency and purity. Cultures derived from genomically stable iPSCs exhibited reduced variance and improved MN marker expression profiles. These findings support routine genomic assessment of iPSCs as a practical and effective strategy to enhance the reliability of iPSC-derived MN models, thereby improving their utility in preclinical MND research and therapeutic development.