Treatment of iPSC-derived NSCs with Shh and FGF8 for 10 days followed by GDNF and BDNF for 3 weeks resulted in an efficient differentiation to dopaminergic neurons as a large numbers of cells expressed TH by immunocytochemistry (Fig. 2A–2C). For both iPSC lines, approximately 30% ± 5% of total cells were positive for TH, which was comprisable to the efficiency seen in hESC-derived NSC differentiation [5]. Importantly, nearly 100% of the TH-expressing neurons coexpressed nigral marker Girk2 (Fig. 2D), indicating that these neurons were of A9 dopaminergic neurons. Expression of additional midbrain and dopaminergic markers in iPSC-derived neurons was also assessed by real-time quantitative PCR. As expected, several markers including En1, Otx2, Lamx1b, Msx1, Nurr1, Lmx1b, Aromatic L-Amino Acid Decarboxylase (AADC), Vesicular Monoamine Transporter (VMAT), and dopamine transporter (DAT) were upregulated in dopaminergic populations compared with the expression in NSCs (Fig. 2E).
