New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

• Klíčová slova
• endothelial differentiation, induced pluripotent stem cells, peripheral blood mononuclear cells,
• MeSH
• biologické markery metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• endoteliální buňky pupečníkové žíly (lidské) cytologie   metabolismus   MeSH
• endoteliální buňky cytologie   metabolismus   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fyziologická neovaskularizace fyziologie   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární cytologie   metabolismus   MeSH
• lidé MeSH
• regenerativní lékařství metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

• Klíčová slova
• 53BP1, DNA double-strand breaks, DNA repair, Human induced pluripotent stem cells, Long-term in vitro culture, γH2AX,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné linie MeSH
• DNA genetika   metabolismus   MeSH
• dvouřetězcové zlomy DNA * účinky záření   MeSH
• exprese genu MeSH
• fibroblasty cytologie   metabolismus   účinky záření   MeSH
• fosforylace účinky záření   MeSH
• histony genetika   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   účinky záření   MeSH
• kontrolní body fáze G1 buněčného cyklu genetika   MeSH
• kontrolní body fáze G2 buněčného cyklu genetika   MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   metabolismus   účinky záření   MeSH
• oprava DNA genetika   MeSH
• přeprogramování buněk MeSH
• stárnutí buněk genetika   účinky záření   MeSH
• záření gama MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 53BP1 MeSH
• DNA MeSH
• H2AX protein, human MeSH Prohlížeč
• histony MeSH
• TP53BP1 protein, human MeSH Prohlížeč

The incorporation of histone H3 with an acetylated lysine 56 (H3K56ac) into the nucleosome is important for chromatin remodeling and serves as a marker of new nucleosomes during DNA replication and repair in yeast. However, in human cells, the level of H3K56ac is greatly reduced, and its role during the cell cycle is controversial. Our aim was to determine the potential of H3K56ac to regulate cell cycle progression in different human cell lines. A significant increase in the number of H3K56ac foci, but not in H3K56ac protein levels, was observed during the S and G2 phases in cancer cell lines, but was not observed in embryonic stem cell lines. Despite this increase, the H3K56ac signal was not present in late replication chromatin, and H3K56ac protein levels did not decrease after the inhibition of DNA replication. H3K56ac was not tightly associated with the chromatin and was primarily localized to active chromatin regions. Our results support the role of H3K56ac in transcriptionally active chromatin areas but do not confirm H3K56ac as a marker of newly synthetized nucleosomes in DNA replication.

• Klíčová slova
• Cell cycle, Chromatin, DNA replication, H3K56ac, Mammalian cells, Nucleosome,
• MeSH
• buněčný cyklus genetika   fyziologie   MeSH
• chromatin metabolismus   MeSH
• G2 fáze genetika   MeSH
• histony metabolismus   MeSH
• HL-60 buňky MeSH
• hmotnostní spektrometrie MeSH
• lidé MeSH
• nukleozomy metabolismus   MeSH
• replikace DNA genetika   fyziologie   MeSH
• S fáze genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• histony MeSH
• nukleozomy MeSH
• Saccharomyces cerevisiae - proteiny 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