Signaling Through Girdin Underlies Excessive Cell Morphogenesis Resulting from Depletion of Neurodevelopmental Disorder-Related Neurexin-2.

Induced neurons (iNeurons) generated via the forced expression of Neurogenin-2 (NGN2) in human pluripotent stem cells (hPSC) represent a highly efficient and robust model system for studying human neuron maturation and function. NGN2-mediated differentiation allows for the rapid conversion of hPSCs or fibroblasts into functionally mature excitatory neurons (iGluNeurons) within a remarkably short timeframe []. This approach bypasses the need for complex, multi-step differentiation protocols and overcomes many of the limitations associated with extrinsic factor-based differentiation, such as variability in neuronal subtype specification and low yield [, ]. As a result, iGluNeurons have gained widespread adoption in neurodevelopmental and neurodegenerative research, particularly for disease modeling, drug screening, and potential therapeutic applications. Despite its widespread use, standardization of culture conditions remains a major challenge. A systematic review of the literature, across 54 peer-reviewed studies, revealed that current NGN2-based differentiation protocols exhibit several differences that can introduce substantial variability in neuronal maturation, network formation, and functional output [–]. Such inconsistencies hinder cross-study comparisons and limit result reproducibility, thereby affecting the interpretation of experimental outcomes. Since induced neuronal cultures are frequently used for pathophysiological studies and high-throughput drug screening, the establishment of optimized and standardized culture conditions is imperative. Among the various culture parameters, three factors appear to have a key role in determining the efficiency of neuronal differentiation and functional maturation: (1) extracellular matrix coating, (2) initial plating density, and (3) cell medium. Differences in these parameters can significantly influence neuronal maturation and electrophysiological properties, and thereby the interpretation of the experimental results. To address this issue, we conducted a systematic comparison of eight distinct combinations of coating, cell density and medium conditions, evaluating their effects on neuronal morphology, proteomic profile, development, and functional maturation. We found that, while substrate coating had minimal impact, both medium composition and plating density strongly influenced the maturation dynamics and neuronal activity. The standardized combinations of culture conditions enable a balance between rapid maturation and long-term functional stability that can be useful to highlight specific disease-related phenotypes. These results provide a benchmark for the design of human neuronal models for brain diseases and high-throughput drug screening.