Maintenance of human embryonic stem cells (hESCs) with stable genome is important for their future use in cell replacement therapy and disease modeling. Our understanding of the mechanisms maintaining genomic stability of hESC and our ability to modulate them is essential in preventing unwanted mutation accumulation during their in vitro cultivation. In this study, we show the DNA damage response mechanism in hESCs is composed of known, yet unlikely components. Clustered oxidative base damage is converted into DNA double-strand breaks (DSBs) by base excision repair (BER) and then quickly repaired by ligase (Lig)3-mediated end-joining (EJ). If there is further induction of clustered oxidative base damage by irradiation, then BER-mediated DSBs become essential in triggering the checkpoint response in hESCs. hESCs limit the mutagenic potential of Lig3-mediated EJ by DNA break end protection involving p53 binding protein 1 (53BP1), which results in fast and error-free microhomology-mediated repair and a low mutant frequency in hESCs. DSBs in hESCs are also repaired via homologous recombination (HR); however, DSB overload, together with massive end protection by 53BP1, triggers competition between error-free HR and mutagenic nonhomologous EJ.-Kohutova, A., Raška, J., Kruta, M., Seneklova, M., Barta, T., Fojtik, P., Jurakova, T., Walter, C. A., Hampl, A., Dvorak, P., Rotrekl, V. Ligase 3-mediated end-joining maintains genome stability of human embryonic stem cells.

• Klíčová slova
• 53BP1, PARP1, alternative DNA end-joining, base excision repair, pluripotent stem cells,
• MeSH
• DNA-ligasa ATP genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA účinky záření   MeSH
• homologní rekombinace MeSH
• kultivované buňky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   fyziologie   MeSH
• nestabilita genomu * MeSH
• oprava DNA spojením konců fyziologie   účinky záření   MeSH
• oprava DNA fyziologie   účinky záření   MeSH
• proteiny vázající poly-ADP-ribosu genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-ligasa ATP MeSH
• LIG3 protein, human MeSH Prohlížeč
• proteiny vázající poly-ADP-ribosu MeSH

INTRODUCTION: Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. MATERIALS AND METHODS: We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. RESULTS: Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. CONCLUSION: Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

• Klíčová slova
• cell differentiation, ferrites, magnetic resonance imaging, neural precursors, superparamagnetic iron oxide nanoparticles,
• MeSH
• buněčná diferenciace * MeSH
• fibroblasty cytologie   MeSH
• imunoenzymatické techniky MeSH
• indukované pluripotentní kmenové buňky cytologie   MeSH
• kontrastní látky chemie   MeSH
• kultivované buňky MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• lysin chemie   MeSH
• magnetická rezonanční tomografie metody   MeSH
• magnetické nanočástice chemie   MeSH
• neurony cytologie   MeSH
• plíce cytologie   MeSH
• plod cytologie   MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kontrastní látky MeSH
• lysin MeSH
• magnetické nanočástice MeSH

The genomic destabilization associated with the adaptation of human embryonic stem cells (hESCs) to culture conditions or the reprogramming of induced pluripotent stem cells (iPSCs) increases the risk of tumorigenesis upon the clinical use of these cells and decreases their value as a model for cell biology studies. Base excision repair (BER), a major genomic integrity maintenance mechanism, has been shown to fail during hESC adaptation. Here, we show that the increase in the mutation frequency (MF) caused by the inhibition of BER was similar to that caused by the hESC adaptation process. The increase in MF reflected the failure of DNA maintenance mechanisms and the subsequent increase in MF rather than being due solely to the accumulation of mutants over a prolonged period, as was previously suggested. The increase in the ionizing-radiation-induced MF in adapted hESCs exceeded the induced MF in nonadapted hESCs and differentiated cells. Unlike hESCs, the overall DNA maintenance in iPSCs, which was reflected by the MF, was similar to that in differentiated cells regardless of the time spent in culture and despite the upregulation of several genes responsible for genome maintenance during the reprogramming process. Taken together, our results suggest that the changes in BER activity during the long-term cultivation of hESCs increase the mutagenic burden, whereas neither reprogramming nor long-term propagation in culture changes the MF in iPSCs.

• MeSH
• buněčná diferenciace účinky záření   MeSH
• buněčné linie MeSH
• genetické lokusy * MeSH
• hypoxanthinfosforibosyltransferasa genetika   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• lidé MeSH
• mutační rychlost * MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hypoxanthinfosforibosyltransferasa MeSH