Genome stability of human induced pluripotent stem cells

ŘEHÁKOVÁ, Daniela

Human induced pluripotent stem cells (hiPSCs) play roles in both disease modeling and regenerative medicine. It is critical that the genomic integrity of the cells remains intact and that the DNA repair systems are fully functional. I focused on the detection of DNA double-strand breaks (DSBs) by phosphorylated histone H2AX (known as H2AX) and p53-binding protein 1 (53BP1) in multiple lines of hiPSCs, their source cells, human embryonic stem cells (hESCs) and cells differentiated from hiPSCs. I measured spontaneously occurring DSBs throughout the process of reprogramming and during long-term in vitro culture and differentiation process. To assess the variations in the functionality of the DNA repair system among the samples, the number of DSBs induced by gamma irradiation and the decrease over time was analyzed. The foci number was detected by fluorescence microscopy separately for the G1 and S/G2 cell cycle phases. Source cells contained a low number of non-replication-related foci, while this number increased after reprogramming into hiPSCs and then decreased again after long-term in vitro passaging or differentiation. 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. Observations suggest that cellular reprogramming increases the DSB number but that the repair mechanism functions well. However, with long-term hipSCs culture reparation capacity decreases.

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