A ROADMAP FOR INVESTIGATING FUNCTIONALLY MATURE HUMAN GLUTAMATERGIC NEURONS AND THEIR NEURONAL NETWORKS IN EPILEPSY MODELS

Neurological disorders affect 3.4 billion people worldwide (43% of the global population), with projections of nearly 5 billion cases by 2050 due to aging populations and environmental risk factors. This crisis has driven development of scalable, human-relevant 2D/3D in vitro models recapitulating key features disrupted in epilepsy and neurodegeneration, such as synaptic connectivity and excitatory-inhibitory balance.
We generated human-induced glutamatergic neurons (iGluNeurons) via Neurogenin-2 (NGN2) overexpression in pluripotent stem cells, optimized by FACS sorting and pre-differentiation for >95% purity (Servetti et al., 2025). Despite widespread use in disease modeling, NGN2 protocols lack standardization of critical variables -coating substrates, plating density, and culture media- that profoundly impact differentiation efficiency and functional maturation. We systematically reviewed 54 peer-reviewed studies to identify optimal parameters, then tested combinations of two coatings (poly-L-ornithine, polyethyleneimine), two media (BrainPhys [BP], Neurobasal [NB]), and two densities (high: 4800 cells/mm²; low: 1200 cells/mm²).
Multi-electrode array recordings, patch-clamp (single-cell/network), and proteomics across developmental timepoints revealed media composition as the dominant factor. Low-density cultures outperformed high-density, while NB media supported sustained network maturation versus BP's rapid-but-declining activity. NB enhanced expression of glutamate receptors, adhesion molecules and synaptic proteins.
We extended this framework to patient-derived iGluNeurons from PRRT2 (paroxysmal kinesigenic dyskinesia/infantile epilepsy), and gene-edited PRRT2 knockout lines with their isogenic controls. Both patient-derived and PRRT2 knockout iGluNeurons displayed significant alterations in firing properties compared with controls. Collectively, these findings establish a robust and standardized framework for iGluNeuron cultures, enabling the reliable detection of disease-relevant functional differences for neurodevelopmental research and disease modeling.