H1 human embryonic stem cells (hES, Wi Cell, Madison, WI) were cultured using standard protocols on inactivated mouse embryonic fibroblasts. Differentiation of hES cells to dopamine neurons was done as described previously43. Human ES cells were cultured on matrigel (Corning, CB-40234)-coated plates at a density of 40,000 cells/cm2 in SRM media containing growth factor and small molecule including FGF8a (100ng/ml, R&D Systems, 423-F8-025/CF), SHH C25II (100ng/ml, R&D Systems, 464-SH-025/CF), LDN193189 (100nM, Stemgent Inc., 04-0074-02), SB431542 (10μM, Stemgent Inc., 04-0010), CHIR99021 (3μM, Stemgent Inc., 04-0004-02) and Purmorphine (2μM, Stemgent Inc., 04-0009) for first five days. For the next six days’ cells were maintained in neurobasal medium containing B27 minus vitamin A (Life Technologies, 12587-010), N2 supplement (Life Technologies, 17502048) along with LDN193189 and CHIR99021. In the final stage cultures were lifted and replated at a density of 400,000/cm2 on polyornithine- and laminin-coated plate in a neurobasal media containing B27 minus Vitamin A, BDNF (20ng/ml, R&D Systems, 248-BD-025/CF), GDNF (20ng/ml, R&D Systems, 212-GD-010/CF), TGFβ (1ng/ml, R&D Systems, 243-B3-002/CF) ascorbic acid (0.2mM, Sigma, A4034), cAMP (0.5mM, Sigma D0627) and DAPT (10μM, Stemgent, 04-0041) till maturation. Once mature (approximately 60 days) toxicity assays with A1 ACM were performed as outlined above.
