Abstract
Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson’s disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.
Highlights
Ethics approval and consent to participate. iPSCs were derived from donors who had given signed informed consent for derivation of iPSC lines from skin biopsies as part of the EU IMI-funded programme StemBANCC
This high content method could be used to test a number of potential therapeutics with the aim of discovering compounds that can rescue mitochondrial defects in PD patient cells and could be applied to testing potential therapeutics for other neurodegenerative diseases associated with mitochondrial dysfunction
Summary
IPSCs were maintained on Matrigel in Essential 8 medium and passaged with collagenase IV. Fibroblasts were cultured in DMEM with l-glutamine and passaged with trypsin-EDTA. IPSCs were differentiated into midbrain dopaminergic neurons as previously described[79]. Cells were plated at high density on Matrigel in iPSC media, transferred to neuronal induction media consisting of knockout DMEM with knockout serum replacement supplemented with LDN193189, SB431542, sonic hedgehog C24II, FGF8A, Purmorphamine + CHIR99021. Cells were passaged using Accutase after 21 days, and seeded onto plates coated with poly-L-ornithine, laminin and fibronectin. Cells were passaged approximately 3 more times before being seeded in 384 well plates. Cells were frozen at day 35 and later thawed, grown for approximately another two weeks before being seeded in 384 well plates
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