Radiation Response of Murine Embryonic Stem Cells

Christine E Hellweg,Claudia Schmitz,Margit Henry,Sebastian Feles,Tamara Rotshteyn,Vaibhav Shinde,Luis F Spitta,Christa Baumstark-Khan,Ruth Hemmersbach,Sureshkumar Perumal Srinivasan,Agapios Sachinidis,Jürgen Hescheler

doi:10.3390/cells9071650

Christine E Hellweg, Claudia Schmitz + Show 10 more

Open Access

https://doi.org/10.3390/cells9071650

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Journal: Cells	Publication Date: Jul 9, 2020
Citations: 8	License type: CC BY 4.0

Affiliation: German Aerospace Center, University of Cologne

Abstract

To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.

Highlights

The genomic integrity of embryonic stem cells is crucial for the fate of embryogenesis
X- irradiation of murine CGR8 embryonic stem cells (mESCs) resulted in DNA double strand breaks, cell cycle arrest in the G2 phase for 16 h after irradiation and cell mortality in a dose-dependent manner and those were independent of leukemia inhibitory factor (LIF)’s presence or absence
Of particular interest were seven developmental genes, which were negatively regulated by X-ray irradiation

Summary

Introduction

The genomic integrity of embryonic stem cells is crucial for the fate of embryogenesis. Embryonic stem cells (ESCs) have the potential to differentiate into several cell types and to self-renew [1], allowing. Ionizing radiation is known to interfere with the genomic integrity of ESCs, which may hamper the proper embryogenesis [4] and mutations could enter the germline. DNA damage response encompasses cell cycle arrest and DNA repair; failure of these processes can result in cellular senescence or apoptosis. The DNA damage response of ESCs is described to be robust [5]. During the error-prone process of DNA damage repair, various mutations can arise after exposure to ionizing radiation in different types of cells [7,8]

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