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Autophagy pp 257-280 | Cite as

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

  • Petros Stathakos
  • Natalia Jimenez-Moreno
  • Lucy Crompton
  • Paul Nistor
  • Maeve A. Caldwell
  • Jon D. LaneEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

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.

Key words

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

Abbreviations

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Petros Stathakos
    • 1
  • Natalia Jimenez-Moreno
    • 1
  • Lucy Crompton
    • 1
  • Paul Nistor
    • 2
  • Maeve A. Caldwell
    • 3
  • Jon D. Lane
    • 1
    Email author
  1. 1.Cell Biology LaboratoriesSchool of Biochemistry, University of BristolBristolUK
  2. 2.School of Clinical Medicine, University of BristolBristolUK
  3. 3.Trinity College Institute for Neuroscience, Trinity CollegeDublinIreland

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