We present a robust and simple hPSC differentiation protocol to derive a low-density culture of cortical glutamatergic and GABAergic neurons, following in vivo developmental processes. Using electrophysiology, calcium imaging, confocal microscopy, mRNA and proteome analysis, we proved functional activity of excitatory-inhibitory networks with reproducible synaptic balance. Although earlier studies produced mixed neuronal cultures, we generated a low-density network with purely cortical neurons. These cultures facilitate the analysis of inhibitory and excitatory network changes in patient hiPSC-derived cortical networks at single cell resolution.
