An electrophysiological and proteomics roadmap for human induced glutamatergic neurons: fine-tuning of culture conditions for pathophysiological studies

: Induced glutamatergic neurons (iGluNeurons) generated by Neurogenin-2 (NGN2) overexpression in human pluripotent stem cells are a powerful model for studying human neuronal maturation and function; however, NGN2-based protocols still lack standardized culture conditions that critically affect neuronal development and function. Three key factors have been identified by previous literature, namely the composition of extracellular matrix coating, the initial plating density, and the choice of culture medium, but the differential effects of their combination have not been thoroughly analyzed. Here, we investigated the combinatorial effects of these three variables, testing eight distinct culture conditions resulting from the combinations of two coatings (poly-L-ornithine and polyethyleneimine), two media (BrainPhys and Neurobasal), and two cell densities (4800 and 1200 cells/mm²). We assessed electrophysiological properties at the single-cell and network levels, characterized morphofunctional and proteomic features across multiple developmental stages. Electrophysiological data indicate that medium composition and plating density, rather than substrate coating, determine neuronal maturation dynamics, with BrainPhys and high density promoting rapid but transient maturation while Neurobasal and low density supporting gradual and sustained network development. Morphofunctional analyzes of synapses and the axon initial segment, together with neuronal maturation markers, support an early BrainPhys-driven acceleration of development that is later exceeded by Neurobasal. To enable accurate proteome profiling of the iGluNeuron system-comprising human neurons and rat astrocytes-we developed a robust taxonomic filtering algorithm that selectively identifies human-specific proteins. This approach confirmed the presence of a conserved core of NGN2-driven differentiation pathways across all settings, in addition to condition-specific signatures. Finally, in the optimal conditions identified through our experimental analyzes, robust spontaneous and evoked synaptic activity was observed. These results provide a framework for optimizing iGluNeuron cultures, balancing rapid maturation and long-term functional stability, and establishing a benchmark for human neuronal models in disease research and drug screening.