Imaging Flow Cytometry Quantifies Neural Genome Dynamics.
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. © 2019 International Society for Advancement of Cytometry.
['Michel|Nadine|N|', 'Majumdar|Usnish B|UB|', 'Lannigan|Joanne|J|', 'McConnell|Michael J|MJ|']
D001921:Brain D002454:Cell Differentiation D053903:DNA Breaks, Double-Stranded D004249:DNA Damage D004260:DNA Repair D005434:Flow Cytometry D016678:Genome D006657:Histones D006801:Humans D057026:Induced Pluripotent Stem Cells D057054:Molecular Imaging D009030:Mosaicism D055495:Neurogenesis D059010:Single-Cell Analysis
imaging flow cytometry NPCs neurogenesis γH2AX Tuj1 double strand breaks hiPSCs nestin neuronal differentiation stem cells