Abstract
BackgroundHuman induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. In this article, we focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as γH2AX) and p53-binding protein 1 (53BP1) in three distinct lines of hiPSCs, their source cells, and one line of human embryonic stem cells (hESCs).MethodsWe measured spontaneously occurring DSBs throughout the process of fibroblast reprogramming and during long-term in vitro culturing. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by γ-irradiation and the decrease over time was analysed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases.ResultsWe demonstrated that fibroblasts contained a low number of non-replication-related DSBs, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging. The artificial induction of DSBs revealed that the repair mechanisms function well in the source cells and hiPSCs at low passages, but fail to recognize a substantial proportion of DSBs at high passages.ConclusionsOur observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, after prolonged in vitro culturing of hiPSCs, the repair capacity decreases.
Highlights
Human induced pluripotent stem cells play roles in both disease modelling and regenerative medicine
We have found that irradiated highpassage Human induced pluripotent stem cell (hiPSC) load their DNA with much lower amounts of γH2AX and p53-Binding protein 1 (53BP1) than irradiated Human dermal fibroblast (hDF) and lowpassage hiPSCs, suggesting that high-passage hiPSCs are somewhat less proficient at initiating DNA repair
Our results indicate that spontaneously occurring Double-strand break (DSB) are best recognized by both markers in hiPSCs at low passage, while fewer foci were observed in hiPSCs at high passage and in source fibroblasts
Summary
Human induced pluripotent stem cells (hiPSCs) play roles in both disease modelling and regenerative medicine. Human induced pluripotent stem cells (hiPSCs) hold great promise for clinical applications because of their potential to differentiate into all three embryonic germ layers [1,2,3]. To use hiPSCs in cell therapy or disease modelling [4], it is fundamental that they possess an intact genome. Much research has been performed in the field of genome maintenance in mouse and human embryonic stem cells (hESCs). Simara et al Stem Cell Research & Therapy (2017) 8:73 is probably the main cause of the accumulation of genomic instabilities, resulting from the adaptation to culture conditions and clonal selection during passaging. In their study [7], prolonged in vitro culturing was associated with oncogene duplication
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