C25 2 n

Three mouse ESC lines were used: E14tg2a, Pitx3-GFP21 (link) and Lmx1a-GFP8 (link)18 (link). Mouse ESCs were maintained in 0.1% gelatin coated flasks with GMEM supplemented with 10% FCS, NEAA, L-Glutamine, Sodium Pyruvate, 2-mercaptoethanol and LIF and passaged every other day. For monolayer neural differentiation, cells were plated at low density (7,500 cells cm²) in 50:50 ratio of DM/F12 supplemented with N2 and neurobasal supplemented with retinol-free B27 (referred to as N2B27 media). Cultures were then exposed to MEK inhibitor PD0325901 (1 μM, Axon) from day 2–4 and followed by Shh (200 ng/ml, C25 II-N, R&D) and FGF8b (100 ng/ml, Peprotech) from day 6–10. Media was changed every other day and at day 6 of differentiation, cells were split 1:3, replated into Poly-D-lysine and laminin-coated dishes, and maintained in N2B27 media8 (link).

H7 hESCs were maintained in BD Matrigel-coated plates in mTesR1 medium (StemCell Technologies) and passaged every 3–4 days. Monolayer neuronal differentiation was performed to induce cortical glutamatergic and midbrain dopaminergic neurons, respectively, according to published literatures8 (link)29 (link). Briefly, hESCs were subjected to dual SMAD inhibition with SB431542 (10 uM, Tocris) from day 0 to day 3, LDN193189 (100 nM, Tocris) and Dorsomorphin (200 nM, Tocris) from day 0 to day10. For cortical differentiation, cultures were then maintained in N2B27 media until the end of differentiation without additional patterning molecules. For generating dopamine neurons, cells were exposed to the MEK inhibitor PD0325901 (1 mM, Axon) between days 3–7, followed by Shh (200 ng/ml, C25 II-N, R&D), Purmorphamine (1 μM, Tocris) and FGF8b (100 ng/ml, Peprotech) from day 7 to day 11. Postmitotic dopaminergic neurons were maintained in N2B27 media supplemented with BDNF (10 ng/ml Peprotech), GDNF (10 ng/ml, Peprotech) and AA (0.2 mM, Sigma). At days 10 and 20, cells were passaged on Fibronectin and Poly-d-lysine/laminin-coated dishes, respectively.