To study the functional consequences of the N398 genetic variant of CHRNA5, we used each of the six subject-derived iPSC lines to generate human DA neuron cultures resembling midbrain VTA DA neurons, which are critical mediators of the mesolimbic DAergic system, a pathway whose performance is altered in addiction3132. iPSC were differentiated using the dual SMAD inhibition protocol26. Expression of DA-specific and mature neuronal markers was confirmed by ICC staining of the rate-limiting enzyme tyrosine hydroxylase (TH), the early neuronal marker TuJ1 (βIII tubulin, not shown), and the more mature neuronal marker MAP2 (Fig. 1C,D). More than 75% of MAP2-labeled cells coexpressed TH (D398 75.0% ± 0.092, n = 3; N398 78.4% ± 0.027, n = 3). Cells positive for TH were also associated with punctate staining for the vesicular DA transporter (DAT; Fig. 1E,F). To confirm that TH+ cells were postmitotic, cultures were incubated with EdU for 24 hours to label cells in S phase. Only a small fraction of TH+ cells stained positive for EdU (Fig. 1G,H; 3.0 ± 0.007% for D398; 2.02 ± 0.008% for N398, n = 3), indicating that nearly all cells were postmitotic.
