To generate complex neuronal networks representative of in vivo cortical brain circuitries both inhibitory interneurons and excitatory projection neurons should be present. To induce interneuron cell fate, we treated the high-density hPSC-derived NES cells with SHH and VPA. Previous studies showed that SHH ventralizes the neural tube along the anterior-posterior neural axis [29] and thereby induces cortical interneuron specification [44], whereas VPA administration to human and rodent PSC cultures increases the expression of the GABA-synthesizing enzyme GAD65/67 [20, 31]. To induce pure cultures of hPSC-derived GABAergic neurons, Ma and colleagues used extended treatments with SHH (200ng/ml) for 14 days and VPA (10 μM) for 7 days [20]. Since we aimed for induction into ventral interneuron cell fate in only a subset of the progenitors, we tested short-treatment procedures with SHH (400 ng/ml for 4 days) and retained dorsal identities to 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
