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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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).
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):E24CrossRefGoogle Scholar
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):705–718CrossRefGoogle Scholar
Technologies L (2015) Culturing pluripotent stem cells (PSCs) in essential 8TM mediumGoogle Scholar
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):4363–4374CrossRefGoogle Scholar
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):703–714CrossRefGoogle Scholar
Kriks S et al (2011) Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease. Nature 480(7378):547–551CrossRefGoogle Scholar
Ozair MZ, Kintner C, Brivanlou AH (2013) Neural induction and early patterning in vertebrates. Wiley Interdiscip Rev Dev Biol 2(4):479–498CrossRefGoogle Scholar
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):275–280CrossRefGoogle Scholar
Arenas E, Denham M, Villaescusa JC (2015) How to make a midbrain dopaminergic neuron. Development 142(11):1918–1936CrossRefGoogle Scholar
Sullivan GJ et al (2010) Induced pluripotent stem cells: epigenetic memories and practical implications. Mol Hum Reprod 16(12):880–885CrossRefGoogle Scholar
Klionsky DJ et al (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12(1):1–222CrossRefGoogle Scholar
Allan VJ (ed) (2000) Protein localization by fluorescence microscopy. A Practical Approach. Oxford University Press, OxfordGoogle Scholar
Allen GF et al (2013) Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep 14(12):1127–1135CrossRefGoogle Scholar
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 61:139–149CrossRefGoogle Scholar
Betin VM et al (2012) A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress. Autophagy 8(4):664–676CrossRefGoogle Scholar