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Autophagy pp 257–280Cite as

Imaging Autophagy in hiPSC-Derived Midbrain Dopaminergic Neuronal Cultures for Parkinson’s Disease Research

Part of the Methods in Molecular Biology book series (MIMB,volume 1880)


To appreciate the positive or negative impact of autophagy during the initiation and progression of human diseases, the isolation or de novo generation of appropriate cell types is required to support focused in vitro assays. In human neurodegenerative diseases such as Parkinson’s disease (PD), specific subsets of acutely sensitive neurons become susceptible to stress-associated operational decline and eventual cell death, emphasizing the need for functional studies in those vulnerable groups of neurons. In PD, a class of dopaminergic neurons in the ventral midbrain (mDANs) is affected. To study these, human-induced pluripotent stem cells (hiPSCs) have emerged as a valuable tool, as they enable the establishment and study of mDAN biology in vitro. In this chapter, we describe a stepwise protocol for the generation of mDANs from hiPSCs using a monolayer culture system. We then outline how imaging-based autophagy assessment methodologies can be applied to these neurons, thereby providing a detailed account of the application of imaging-based autophagy assays to human iPSC-derived mDANs.

Key words

  • Autophagy
  • hiPSC
  • Stem cells
  • Dopaminergic neurons
  • Cell culture
  • Immunofluorescence
  • Parkinson’s disease

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  • DOI: 10.1007/978-1-4939-8873-0_17
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Brain derived neurotrophic factor


Bovine serum albumin


Cyclic adenosine monophosphate


N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester


Dulbecco’s modified Eagle’s medium


Dimethyl sulfoxide


Glial-derived neurotrophic factor


4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride


Nonessential amino acids




4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate


Sonic hedgehog


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This work is supported by a Parkinson’s UK project grant (G1402), a Wellcome Trust Ph.D. studentship awarded to NJM through the Dynamic Cell Biology program (grant number 083474), and a Medical Research Council Ph.D. studentship (to PS).

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Correspondence to Jon D. Lane .

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Stathakos, P., Jimenez-Moreno, N., Crompton, L., Nistor, P., Caldwell, M.A., Lane, J.D. (2019). Imaging Autophagy in hiPSC-Derived Midbrain Dopaminergic Neuronal Cultures for Parkinson’s Disease Research. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY.

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