DNA double strand breaks detection in pluripotent stem cells with regard to cell cycle stages

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Rok publikování 2016
Druh Konferenční abstrakty
Fakulta / Pracoviště MU

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Popis Human induced pluripotent stem cells (hiPSCs) have a potential in both disease modeling and regenerative medicine. It is of utmost importance that genomic integrity of the cells remains unharmed and DNA reparation systems fully functional. In our research we focus on the detection of DNA double strand breaks (DSBs) by phosphorylated histone H2AX (known as gammaH2AX) and p53-binding protein 1 (53BP1) in fibroblasts, three distinct lines of hiPSCs, and one line of human embryonic stem cells (hESCs). We measured both spontaneously occurred DSBs and DSBs induced by gamma-irradiation and its decrease in time. Foci number was detected by fluorescence microscopy and EdU (5-ethynyl-2-deoxyuridine) was used to discriminate between cell cycle stages. Discrimination between the EdU negative (G1) and positive (S/G2) populations allows excluding the replication-related foci and increase the accuracy of measurement. This is crucial when comparing number of DSBs in cell types with different cell cycle speed (ie. somatic cells and pluripotent cells). EdU discrimination is also valuable when the cell cycle is being modified during experiments in a way that changes proportion of cells in the S/G2 stage (ie. by irradiation or using cell cycle synchronising agents). In EdU negative (G1) group, we detected low number of replication non-related DSBs in fibroblasts, while this number increases significantly after reprogramming into hiPSCs to decrease again after long-term in vitro passaging. However, hiPSCs in high passages responded weakly to gamma-irradiation treatment in comparison to hiPSCs in low passage number, suggesting their DSB-reparation capacity may be compromised.
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