Establishment of a Human Induced Pluripotent Stem Cell-Derived Cerebral Cortex Organoid Model for Neurotoxicity Assessment.

Human pluripotent stem cell (hPSC)-derived brain organoids have emerged as innovative models for drug screening and cytotoxicity evaluation. However, their inherent cellular heterogeneity presents challenges in isolating targeted neuronal populations, such as upper motor neurons, which are crucial for motor cortex function. In this study, we developed motor cortex-like organoids enriched with excitatory glutamatergic and inhibitory GABAergic neurons to assess neurotoxicity in the upper motor neurons-a key component of voluntary motor control. By optimizing the differentiation protocols, we achieved robust expression of in excitatory neurons and in inhibitory neurons by day 30 of the differentiation. The organoids were generated by co-culturing progenitor cells during the early differentiation phase, followed by lineage-specific maturation. Comparative analyses demonstrated that these organoids more accurately recapitulate the human cortical architecture than traditional neural cell line (SK-N-SH neuroblastoma cells). We observed that measures of cell viability and integrity-assessed via cleaved caspase-3 levels, growth-associated protein 43 (), and autophagy-related protein 5 ()-were significantly higher in 3D organoid cultures compared to conventional 2D systems. In toxicological assays, the motor cortex-like organoids exhibited a dose-dependent response to both toxic and non-toxic compounds, highlighting their potential as high-fidelity neurotoxicity screening models. Our findings suggest that hPSC-derived motor cortex-like organoids serve as a robust, physiologically relevant model that can replace animal models in toxicity assessments, offering enhanced accuracy in evaluating compounds that impact the motor cortex while reflecting better human brain physiology.