achieve spontaneous differentiation of glutamatergic neurons as well. To confirm generation of GABAergic and glutamatergic neurons following in vivo development [3, 44], we performed RNA expression profile analyses at different time points of differentiation and immunocytochemistry analysis at the end stage. In our neuronal cultures, the interneuron population consists of CGE- and LGE-derived cells as indicated by expression of post-mitotic markers PROX1 and MEIS2. The cultures also express MGE markers SATB1 and LHX6, but lack MGE marker NKX2.1. Since our RNA profiling analysis did not detect NKX2.1, we also did not expect Parvalbumin (PV)- and Somatostatin (STT)-positive neurons, as previous studies showed that PV and STT is reduced in Nkx2.1 KO mice [45]. We found expression of other GE lineage interneuron markers, like Calbindin in both direct and indirect contact co-cultures. The excitatory neurons were representatives of upper and deep neocortical layer cells, as shown by expression of the post-mitotic markers CUX1, SATB2, TBR1 and CTIP2, respectively. We successfully generated and characterized a protocol to produce a mixture of both cortical glutamatergic and GABAergic neurons in line with in vivo development.
