SNCA Triplication iPSC-Neuron Differentiation

Lymphoblast cell lines were acquired from the Coriell Institute for Medical Research, Line # GM15010, female origin (New Jersey, USA) from a patient carrying a triplication in the SNCA gene, reprogrammed into induced pluripotent stem cells (iPSCs) called line ‘3x-1’, and characterized previously (Stojkovska et al., 2022 (link)). iPSCs were cultured on Matrigel (Corning, #354277) coated plates and maintained in mTESR1 media. Differentiation into midbrain dopaminergic neurons occurred using previously established protocols (Kriks et al., 2011 (link)). Briefly, iPSC lines were accutased (Corning, # 25058CI) and seeded onto Matrigel (Corning, #354277) coated plates, allowed to grow to confluency, then treated with dual SMAD inhibitors followed by a cocktail of growth factors (Cuddy et al., 2019 (link)). After differentiation, the neurons were cultured in neurobasal medium (ThermoFisher, # 21103049) with NeuroCult SM1 Neuronal Supplement (Stem cell technologies, #5711) and 1% glutamine and penicillin/streptomycin. Patient derived SNCA triplication IPSC-neurons were used to evaluate the effects of HKL in a human relevant model of synucleinopathy. Neurons were matured for 60 days and subsequently treated with 10 μM HKL or 0.01% DMSO for 72 h. Cells were pelleted, snap frozen, and shipped to Mayo Clinic-Jacksonville for αsyn and SNCA mRNA quantitation.