of mixed neurons (Fig 5N and 5P), which showed increased amplitude of response to an increasing number of stimuli (Fig 5Q). We also observed ROIs, which upon bicuculline addition showed either increased (20%) or decreased (55%) frequency of activity, and upon bicuculline/AP5/DNQX addition showed persistent activity (10%) and did not recover activity after TTX wash out (17%). Finally, to study differences between direct and indirect contact cultures, we analyzed RNA of both neuronal cultures at end stages for lineage markers of GABAergic and glutamatergic neurons. There are no significant differences in the expression of markers between direct and indirect contact cultures (Fig 3A–3H). Although we need to consider that the end-stage analysis of direct and indirect cultures was at different time points (day 49 and day 56). These observations confirm that hPSC-derived neurons also form a functional and mixed synaptic network of GABAergic and glutamatergic neurons, when grown with rodent astrocytes in an indirect contact co-culture system.
