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Cytometry A. 2019 May 7. doi: 10.1002/cyto.a.23783. [Epub ahead of print]

Imaging Flow Cytometry Quantifies Neural Genome Dynamics.

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Department of Biochemistry & Molecular Genetics, University of Virginia School of Medicine, Neuroscience Graduate Program, Charlottesville, Virginia, 22908.
Department of Health System Design and Global Health, Icahn School of Medicine, 1 Gustave L. Levy Pl, New York, NY 10029.
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia School of Medicine, Flow Cytometry Core Facility, 1340 Jefferson Park Ave., Pinn Hall, Room 2011, Charlottesville, Virginia, 22908-0734.
Department of Neuroscience, University of Virginia School of Medicine, Neuroscience Graduate Program, Charlottesville, Virginia, 22908.


Somatic mosaicism is a common consequence of normal development. DNA repair is simply not perfect, and each cell's genome incurs continuous DNA damage as a consequence of transcription, replication, and other cell biological stressors. Brain somatic mosaicism is particularly noteworthy because the vast majority of an individual's neurons are with that individual for life and neural circuits give rise directly to behavioral phenotypes. Brain somatic mosaicism, now revealed and tractable due to advances in single cell 'omic approaches, has emerged as an intriguing and unexplored aspect of neuronal diversity. Furthermore, the study of DNA damage during early neurodevelopment, when the rate of mutagenesis is high, is the perfect starting point to understand the origins of brain mosaicism. Flow cytometry is a highly efficient technique to study cell cycle and intracellular proteins of interest, particularly those related to DNA damage, but it lacks the high resolution of microscopy to examine the localization of these proteins. In this study, we outline a novel single-cell approach to quantify DNA double-strand break (DNA DSB) dynamics during early human neurodevelopment by applying imaging flow cytometry (IFC) to human-induced pluripotent stem cell-derived neural progenitor cells (NPCs) undergoing neurogenesis. We establish an increase of DNA DSBs by quantifying γH2AX foci in mildly stressed NPCs using various single-cell approaches in addition to IFC including fluorescent microscopy, conventional flow cytometry, and measuring DNA DSBs with the comet assay. We demonstrate the dose-dependent sensitive detection of γH2AX foci through IFC and reveal the dynamics of DNA DSBs in proliferating and differentiating neural cells in early neurogenesis.


NPCs; Tuj1; double strand breaks; hiPSCs; imaging flow cytometry; nestin; neurogenesis; neuronal differentiation; stem cells; γH2AX


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