Developing a Neuronal Differentiation Score for quantitative assessment of glutamatergic differentiation in human...

Reliable in vitro neuronal models are essential for studying neuronal development and neurological diseases. Although SH-SY5Y cells, derived from human neuroblastoma, are widely used for neuronal differentiation, their efficiency varies markedly with experimental conditions, and standardized quantitative evaluation methods remain lacking. Here, we systematically optimized glutamatergic differentiation of SH-SY5Y cells by modulating serum concentration, extracellular matrix growth factors, induction duration, and basal medium composition. Reduced serum levels promoted neurite outgrowth, while growth factor-enriched Matrigel and nutrient-rich media enhanced neurite complexity and expression of neuronal markers. Prolonged induction further augmented glutamatergic differentiation. To enable objective and quantitative assessment, we developed a Neuronal Differentiation Score (NDS) by integrating morphological and molecular features via principal component analysis. The NDS strongly correlated with neuronal architecture and marker expression, allowing reliable classification of differentiation grades (high-grade defined as NDS > -0.0967). These findings provide an optimized protocol for reproducible glutamatergic differentiation of SH-SY5Y cells and a validated, quantitative tool for assessing neuronal maturation, facilitating more reliable in vitro modeling of neurodevelopment and disease.