In Vitro Induction of Human Embryonic Stem Cells into the Midbrain Dopaminergic Neurons and Transplantation in Cynomolgus Monkey

A simple, rapid, efficient, and specialized culture system was successfully developed in this study to induce human embryonic stem cells into dopaminergic neurons in vitro. It only took 5 days to generate quickly and directly a large number of homogeneous neural stem cell (NSC) spheres by the introduction of small molecules LDN (inhibitor of BMP [bone morphogenetic protein] pathway that inhibits BMP type I receptors ALK(2) and ALK(3)), SB431542 (inhibitor of TGF-beta/Activin/Dodal pathway that inhibits ALK(4), ALK(5), and ALK(7)), CHIR99021 (inhibitors of GSK-3 [glycogen synthase kinase 3]), and basic fibroblast growth factor (bFGF). The dopaminergic neurons were successfully induced at day 25 (tyrosine hydroxylase [TH] expressed) and at day 32 (TH highly expressed) with high purity (TH/Tuj1: 84.14% and 93.15%, respectively) by the addition of FGF8 (fibroblast growth factor 8), sonic hedgehog (SHH), and Purmorphamine after the generation of NSC at day 5. And, the dopaminergic neurons induced by this system successfully survived and integrated into the striatum of cynomolgus monkey brain after transplantation, which verified the efficiency of the induction system developed in this study, suggesting the potential clinical application in cell therapy for neurological diseases.