Objective.The purpose of this study is to investigate whether and how the balance between excitation and inhibition ('E/I balance') influences the spontaneous development of human-derived neuronal networksin vitro. To achieve that goal, we performed a long-term (98 d) characterization of both homogeneous (only excitatory or inhibitory neurons) and heterogeneous (mixed neuronal types) cultures with controlled E/I ratios (i.e. E:I 0:100, 25:75, 50:50, 75:25, 100:0) by recording their electrophysiological activity using micro-electrode arrays.Approach.Excitatory and inhibitory neurons were derived from human induced pluripotent stem cells (hiPSCs). We realized five different configurations by systematically varying the glutamatergic and GABAergic percentages.Main results.We successfully built both homogeneous and heterogeneous neuronal cultures from hiPSCs finely controlling the E/I ratios; we were able to maintain them for up to 3 months. Homogeneity differentially impacted purely inhibitory (no bursts) and purely excitatory (few bursts) networks, deviating from the typical traits of heterogeneous cultures (burst dominated). Increased inhibition in heterogeneous cultures strongly affected the duration and organization of bursting and network bursting activity. Spike-based functional connectivity and image-based deep learning analysis further confirmed all the above.Significance.Healthy neuronal activity is controlled by a well-defined E/I balance whose alteration could lead to the onset of neurodevelopmental disorders like schizophrenia or epilepsy. Most of the commonly usedin vitromodels are animal-derived or too simplified and thus far from thein vivohuman condition. In this work, by performing a long-term study of hiPSCs-derived neuronal networks obtained from healthy human subjects, we demonstrated the feasibility of a robustin vitromodel which can be further exploited for investigating pathological conditions where the E/I balance is impaired.

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