DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing.
Human 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).



We 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.



We 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.



Our 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.
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic. p.simara@mail.muni.cz.
Pavel
0000-0001-7581-7048
P
Simara
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Lenka
L
Tesarova
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Daniela
D
Rehakova
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Pavel
P
Matula
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Stanislav
S
Stejskal
Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Kamenice 3, 625 00, Brno, Czech Republic.
Ales
A
Hampl
Centre for Biomedical Image Analysis, Faculty of Informatics, Masaryk University, Kamenice 5, 625 00, Brno, Czech Republic.
Irena
I
Koutna
D002460:Cell Line; D065150:Cellular Reprogramming; D016922:Cellular Senescence / Q000235:genetics / Q000528:radiation effects*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D059585:G1 Phase Cell Cycle Checkpoints / Q000235:genetics; D059565:G2 Phase Cell Cycle Checkpoints / Q000235:genetics; D005720:Gamma Rays; D015870:Gene Expression; D006657:Histones / Q000235:genetics / Q000378:metabolism; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D010766:Phosphorylation / Q000528:radiation effects; D000071857:Tumor Suppressor p53-Binding Protein 1 / Q000235:genetics / Q000378:metabolism
C468783:H2AX protein, human; D006657:Histones; C498301:TP53BP1 protein, human; D000071857:Tumor Suppressor p53-Binding Protein 1; D004247:DNA
53BP1; DNA double-strand breaks; DNA repair; Human induced pluripotent stem cells; Long-term in vitro culture; γH2AX