Human ESC (H9, H1) and iPSC lines (2C6 and SeV6) were subjected to a modified dual SMAD-inhibition13 based FP induction12 protocol. Exposure to SHH C25II, Purmorphamine, FGF8 and CHIR99021 were optimized for midbrain FP and DA neuron yield (see Figure 1d). Following FP induction, further maturation was carried out in Neurobasal/B27 medium supplemented with AA, BDNF, GDNF, TGFβ3 and dbcAMP (see full methods for details). The resulting DA neurons were subjected to extensive phenotypic characterization via immunocytochemistry, qRT-PCR, gene expression profiling, HPLC analysis for DA and in vitro electrophysiological recordings. In vivo studies were performed in 6-hydroxydopamine lesioned, hemiparkinsonian rodents (adult NOD-SCID IL2Rgc mice and Sprague Dawley rats) as well as in two adult rhesus monkeys treated with carotid injections of MPTP. DA neurons were injected stereotactically in the striata of the animals (150 × 103 cells in mice, 250 × 103 cells in rats) and a total of 7.5 × 106 cells (distributed in 6 tracts; 3 on each side of brain) in monkeys. Behavioral assays were performed at monthly intervals post grafting, including amphetamine mediated rotational analysis as
