Abstract
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.
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- BDNF:
-
Brain derived neurotrophic factor
- BSA:
-
Bovine serum albumin
- cAMP:
-
Cyclic adenosine monophosphate
- DAPT:
-
N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- DMSO:
-
Dimethyl sulfoxide
- GDNF:
-
Glial-derived neurotrophic factor
- LDN193189:
-
4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline hydrochloride
- NEAA:
-
Nonessential amino acids
- Pen/Strep:
-
Penicillin/Streptomycin
- SB431542:
-
4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate
- SHH:
-
Sonic hedgehog
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Acknowledgments
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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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. https://doi.org/10.1007/978-1-4939-8873-0_17
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DOI: https://doi.org/10.1007/978-1-4939-8873-0_17
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-8873-0
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