A monolayer hiPSC culture system for autophagy/mitophagy studies in human dopaminergic neurons

Autophagy cytoplasmic quality control pathways are required during neural development, and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases including Parkinson’s disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening; and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs)—the predominant neuronal sub-type afflicted in PD—have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability, and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.