Imaging Flow Cytometry Quantifies Neural Genome Dynamics

Abstract

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.

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks

“…HeLa cells (ATCC) and GM13069 cells (ATCC) were grown in DMEM (Gibco) and RPMI 1640 medium (Gibco), respectively, and supplemented with 10% fetal bovine serum. Human neural progenitor cells (NPC9429) derived from an apparently healthy individual ( 41 ), were grown in DMEM/F12 (Gibco) media containing 1% N-2 (Gibco), 2% B27-A (Gibco), 1μg/mL laminin (Life Technology) and fibroblast growth factor (Peprotech), and maintained in matrigel (Corning)-coated dishes. Etoposide (Sigma) treatment of GM13069 cells were at 0.15, 1.5 or 15 μM for 24 h, along with untreated cells.…”

Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis

“…DNA DSB levels were quantified at 6 weeks of hiPSC-based neurogenesis using IFC 45 , 49 , 50 . Immunoreactive γH2aX puncta were counted in single β-III tubulin-positive cells (i.e., neurons) using the TUJ1 antibody 51 .…”

“…Previously, we developed imaging flow cytometry (IFC) 45 to quantify DNA DSBs with single cell resolution during hiPSC-based neurogenesis. This study revealed increasing levels of DNA DSBs during the first 2 weeks of hiPSC-based neurogenesis prompting us to further investigate neurogenic DNA damage signaling.…”

The Phosphorylated Form of the Histone H2AX (γH2AX) in the Brain from Embryonic Life to Old Age

“…In accord with this possibility, the loss of the breast cancer type 2 susceptibility protein (BRCA2), another protein involved in DSB repair and/or HR, results in defective embryonic (and postnatal) neurogenesis [ 100 ]. In addition, a recent cytofluorimetric study has demonstrated that actively replicating neural progenitor cells harbor more DNA DSBs during early neurogenesis than at later differentiation stages [ 101 ]. Moreover, a study on mice lacking recombination activating 2 (RAG-2), a component of the RAG-1,2-complex responsible for initiating somatic recombination in lymphocytes, led to the identification of the long interspersed element-1 (LINE-1) retrotransposition as one of the sources of DSBs in retinal neurogenesis and to the suggestion that both DSBs generation and repair are genuine processes intrinsic to neural development [ 102 ].…”