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Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorders

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Book cover Induced Pluripotent Stem Cells and Human Disease

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2549))

Abstract

Mitochondria are responsible for many vital pathways governing cellular homeostasis, including cellular energy management, heme biosynthesis, lipid metabolism, cellular proliferation and differentiation, cell cycle regulation, and cellular viability. Electron transport and ADP phosphorylation coupled with proton pumping through the mitochondrial complexes contribute to the preservation of mitochondrial membrane potential (ΔΨm). Importantly, mitochondrial polarization is essential for reactive oxygen species (ROS) production and cytosolic calcium (Ca2+) handling. Thus, changes in mitochondrial oxidative phosphorylation (OXPHOS), ΔΨm, and ATP/ADP may occur in parallel or stimulate each other. Brain cells like neurons are heavily reliant on mitochondrial OXPHOS for its high-energy demands, and hence improper mitochondrial function is detrimental for neuronal survival. Indeed, several neurodegenerative disorders are associated with mitochondrial dysfunction. Modeling this disease-relevant phenotype in neuronal cells differentiated from patient-derived human induced pluripotent stem cells (hiPSCs) provide an appropriate cellular platform for studying the disease pathology and drug discovery. In this review, we describe high-throughput analysis of crucial parameters related to mitochondrial function in hiPSC-derived neurons. These methodologies include measurement of ΔΨm, intracellular Ca2+, oxidative stress, and ATP/ADP levels using fluorescence probes via a microplate reader. Benefits of such an approach include analysis of mitochondrial parameters on a large population of cells, simultaneous analysis of different cell lines and experimental conditions, and for drug screening to identify compounds restoring mitochondrial function.

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Acknowledgments

We thank Malkiel Cohen for the neuronal differentiation protocol, BioRender.com via which the figure is created, and the funding agencies for supporting our research. SS has been funded by research grants from LifeArc (P2019-0004), UKIERI (UK-India Education and Research Initiative; 2016-17-0087), Wellcome Trust (109626/Z/15/Z, 1516ISSFFEL10), and Birmingham Fellowship; TRR from FAPESP (São Paulo Research Foundation; 2015/02041-1) and CNPq/CAPES; SS and TRR by FAPESP–Birmingham–Nottingham Strategic Collaboration Fund, Rutherford Fellowship, and University of Birmingham Brazil Visiting Fellowship. SS is also a former fellow for life at Hughes Hall, University of Cambridge, UK.

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Authors and Affiliations

  1. Institute of Cancer and Genomic Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK

    Tatiana R. Rosenstock, Congxin Sun, Georgina Wynne Hughes, Katherine Winter & Sovan Sarkar

  2. Department of Pharmacology, University of São Paulo, São Paulo, Brazil

    Tatiana R. Rosenstock

  3. Department of Bioscience, Sygnature Discovery, BioCity, Nottingham, United Kingdom

    Tatiana R. Rosenstock

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  1. Tatiana R. Rosenstock
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  2. Congxin Sun
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  3. Georgina Wynne Hughes
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  4. Katherine Winter
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  5. Sovan Sarkar
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Correspondence to Sovan Sarkar .

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  1. Ottawa, ON, Canada

    Kursad Turksen

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Rosenstock, T.R., Sun, C., Hughes, G.W., Winter, K., Sarkar, S. (2022). Analysis of Mitochondrial Dysfunction by Microplate Reader in hiPSC-Derived Neuronal Cell Models of Neurodegenerative Disorders. In: Turksen, K. (eds) Induced Pluripotent Stem Cells and Human Disease. Methods in Molecular Biology, vol 2549. Humana, New York, NY. https://doi.org/10.1007/7651_2021_451

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  • DOI: https://doi.org/10.1007/7651_2021_451

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2584-2

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