A New Selection Strategy to Enrich Dopaminergic Neurons Facilitates Comparisons Between Patient iPSC-Derived Lines

Comparative studies of dopaminergic (DA) neurons differentiated from induced pluripotent stem cells (iPSCs) derived from patients discordant for parkinsonism, especially siblings, provide a valuable model to explore factors underlying Parkinson disease (PD) pathogenesis. However, such studies are complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich DA neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene, placing its expression under the endogenous promoter. Using iPSC-derived neurons from a healthy individual (HT711), we achieved selective enrichment of TH+ neurons through geneticin selection without compromising differentiation capacity, as demonstrated by Western blots, immunocytochemistry and flow cytometry. We then applied the method to iPSCs derived from two siblings of HT711, one with Gaucher disease (GD) alone and one with both PD and GD. Quantification of multiple DA neuron markers through whole-cell proteomics confirmed their comparable enrichment in the three lines, demonstrating that the new method can be applied to iPSC lines from different individuals to standardize DA neuron cultures. While we focus on methodological developments here, a companion analysis of the same whole-cell proteomics dataset, examining molecular features potentially associated with disease statuses, has been reported separately.