RESUMO
Patient-specific induced pluripotent stem cells (iPSCs) are a powerful tool to investigate the molecular mechanisms underlying Parkinson's disease (PD), and might provide novel platforms for systematic drug screening. Several strategies have been developed to generate iPSC-derived tyrosine hydroxylase (TH)-positive dopaminergic neurons (DAn), the clinically relevant cell type in PD; however, they often result in mixed neuronal cultures containing only a small proportion of TH-positive DAn. To overcome this limitation, we used CRISPR/Cas9-based editing to generate a human iPSC line expressing a fluorescent protein (mOrange) knocked-in at the last exon of the TH locus. After differentiation of the TH-mOrange reporter iPSC line, we confirmed that mOrange expression faithfully mimicked endogenous TH expression in iPSC-derived DAn. We also employed calcium imaging techniques to determine the intrinsic functional differences between dopaminergic and non-dopaminergic ventral midbrain neurons. Crucially, the brightness of mOrange allowed direct visualization of TH-expressing cells in heterogeneous cultures, and enabled us to isolate live mOrange-positive cells through fluorescence-activated cell sorting, for further differentiation. This technique, coupled to refined imaging and data processing tools, could advance the investigation of PD pathogenesis and might offer a platform to test potential new therapeutics for PD and other neurodegenerative diseases.
