We generated low-density hPSC-derived neuronal cultures of GABAergic-glutamatergic neurons, which are amenable to multi-level analysis from early developmental to functional stages. We performed RNA expression analysis and immunocytochemistry to analyze neuronal and synaptic development, and studied functional properties by calcium imaging and patch-clamp electrophysiology. To support the maturation of neuronal precursors into functional neurons, rat astrocytes were supplemented using either a direct contact or an indirect contact co-culture system. Neuronal cell populations in the indirect co-culture mode showed no expression of glial genes, which gives new tools to study neuronal-specific changes in functional hPSC-derived cultures. These well-characterized low-density cultures will facilitate the study of disease mechanisms underlying neurodevelopmental disorders particularly involving inhibitory and excitatory network changes.
