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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)

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

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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

References

  1. Jimenez-Moreno N et al (2017) Induced pluripotent stem cell neuronal models for the study of autophagy pathways in human neurodegenerative disease. Cell 6(3):E24

    CrossRef  Google Scholar 

  2. Jungverdorben J, Till A, Brustle O (2017) Induced pluripotent stem cell-based modeling of neurodegenerative diseases: a focus on autophagy. J Mol Med (Berl) 95(7):705–718

    CAS  CrossRef  Google Scholar 

  3. Surmeier DJ, Obeso JA, Halliday GM (2017) Selective neuronal vulnerability in Parkinson disease. Nat Rev Neurosci 18(2):101–113

    CAS  CrossRef  Google Scholar 

  4. Technologies L (2015) Culturing pluripotent stem cells (PSCs) in essential 8TM medium

    Google Scholar 

  5. Jaeger I et al (2011) Temporally controlled modulation of FGF/ERK signaling directs midbrain dopaminergic neural progenitor fate in mouse and human pluripotent stem cells. Development 138(20):4363–4374

    CAS  CrossRef  Google Scholar 

  6. Kirkeby A et al (2012) Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions. Cell Rep 1(6):703–714

    CAS  CrossRef  Google Scholar 

  7. Kriks S et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480(7378):547–551

    CAS  CrossRef  Google Scholar 

  8. Ozair MZ, Kintner C, Brivanlou AH (2013) Neural induction and early patterning in vertebrates. Wiley Interdiscip Rev Dev Biol 2(4):479–498

    CAS  CrossRef  Google Scholar 

  9. Chambers SM et al (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27(3):275–280

    CAS  CrossRef  Google Scholar 

  10. Arenas E, Denham M, Villaescusa JC (2015) How to make a midbrain dopaminergic neuron. Development 142(11):1918–1936

    CAS  CrossRef  Google Scholar 

  11. Sullivan GJ et al (2010) Induced pluripotent stem cells: epigenetic memories and practical implications. Mol Hum Reprod 16(12):880–885

    CAS  CrossRef  Google Scholar 

  12. Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222

    CrossRef  Google Scholar 

  13. Allan VJ (ed) (2000) Protein localization by fluorescence microscopy. A Practical Approach. Oxford University Press, Oxford

    Google Scholar 

  14. Allen GF et al (2013) Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep 14(12):1127–1135

    CAS  CrossRef  Google Scholar 

  15. Nistor PA et al (2015) Long-term culture of pluripotent stem-cell-derived human neurons on diamond—a substrate for neurodegeneration research and therapy. Biomaterials 61:139–149

    CAS  CrossRef  Google Scholar 

  16. Betin VM et al (2012) A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress. Autophagy 8(4):664–676

    CAS  CrossRef  Google Scholar 

Download references

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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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. 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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