To determine whether the cultures were capable of releasing DA in response to stimulation, we replaced culture medium with buffer containing 1 mM nicotine for 30 minutes, and analyzed the DA content in the medium using HPLC. A distinct peak consistent with DA retention time was observed (Fig. 1I,J). All results are consistent with the presence of midbrain-like DA neurons, but the cultures also contained a small minority of serotonergic (5-HT positive) and inhibitory (GAD6 positive) neurons (Fig. 1K–N). Results also identified cells that were both TH+ and VGluT+ (Fig. 1O,P), consistent with the notion that some DA neurons also co-release glutamate3334. Moreover, DA cultures express mRNAs encoding nAChR subunits α3, α4, α5, α6, β2, and β4 at levels similar to those found in developing midbrain VTA, using a sample of human fetal VTA mRNA as a reference (Fig. 1Q). To further confirm the lineage of our culture, we observed mRNA expression for TH and the classic midbrain marker, PITX3 (Fig. 1R). All nAChR subunit and marker mRNAs increased significantly upon differentiation from iPSC to midbrain-like DA neuron cultures (ANOVA,
