A Modified Differentiation Protocol In Vitro to Generate Dopaminergic Neurons from Pluripotent Stem Cells

Cell transplantation is considered a promising therapeutic strategy for the treatment of Parkinson's disease. Because of their strong differentiation potential, pluripotent stem cells may become a source of dopaminergic neurons for cell transplantation. Although published protocols have revealed that pluripotent stem cells can be successfully induced into dopaminergic neurons, unwanted cell types still exist in PSC-derived cultures. Therefore, signaling parameters for dopaminergic neuron patterning in differentiation protocols need to be further identified and optimized. In this study, we explored an in vitro modified differentiation protocol for efficiently inducing dopaminergic neurons from pluripotent stem cells. Briefly, pluripotent stem cells were incubated in N2B27 medium for a 4-day culture, and then bFGF, SHH-C24II, purmorphamine, FGF8a and laminin were added to the medium. After a 6-day culture, the medium was replaced with N2B27 medium containing L-ascorbic acid, glial cell line-derived neurotrophic factor, cyclic adenosine monophosphate, laminin, and brainderived neurotrophic factor for an additional 10 days. We confirmed that combined treatment with bFGF, SHH-C24II, purmorphamine, FGF8a and laminin significantly promoted the differentiation of pluripotent stem cells into dopaminergic neurons. Additionally, we determined a reasonable time window for the use of these factors. Our study provides new insights into the role of cell factors in dopaminergic neuron differentiation of pluripotent stem cells.