The work with midbrain dopaminergic neurons (mDAN) differentiation might seem to be hard. There are about 40 different published protocols for mDAN differentiation, which are eventually modified according to the respective laboratory. In many cases, protocols are not fully described, failing to provide essential tips for researchers starting in the field. Considering that commercial kits produce low mDAN percentages (20-50%), we chose to follow a mix of four main protocols based on Kriks and colleagues' protocol, from which the resulting mDAN were engrafted with success in three different animal models of Parkinson's disease. We present a differential step-by-step methodology for generating mDAN directly from human-induced pluripotent stem cells cultured with E8 medium on Geltrex, without culture on primary mouse embryonic fibroblasts prior to mDAN differentiation, and subsequent exposure of neurons to rock inhibitor during passages for improving cell viability. The protocol described here allows obtaining mDAN with phenotypical and functional characteristics suitable for in vitro modeling, cell transplantation, and drug screening.
Keywords: Cell differentiation; Cell reprogramming; Dopaminergic differentiation; Dopaminergic neurons; In vitro modeling; Induced pluripotent stem cells; Neural differentiation; Neurodegenerative disease models; Parkinson’s disease.