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.

• Keywords
• endothelial differentiation, induced pluripotent stem cells, peripheral blood mononuclear cells,
• MeSH
• Biomarkers metabolism   MeSH
• Cell Differentiation physiology   MeSH
• Human Umbilical Vein Endothelial Cells cytology   metabolism   MeSH
• Endothelial Cells cytology   metabolism   MeSH
• Fibroblasts cytology   metabolism   MeSH
• Neovascularization, Physiologic physiology   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   MeSH
• Cells, Cultured MeSH
• Leukocytes, Mononuclear cytology   metabolism   MeSH
• Humans MeSH
• Regenerative Medicine methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers 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.

• Keywords
• 53BP1, DNA double-strand breaks, DNA repair, Human induced pluripotent stem cells, Long-term in vitro culture, γH2AX,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 genetics   metabolism   MeSH
• Cell Line MeSH
• DNA genetics   metabolism   MeSH
• DNA Breaks, Double-Stranded * radiation effects   MeSH
• Gene Expression MeSH
• Fibroblasts cytology   metabolism   radiation effects   MeSH
• Phosphorylation radiation effects   MeSH
• Histones genetics   metabolism   MeSH
• Induced Pluripotent Stem Cells cytology   metabolism   radiation effects   MeSH
• G1 Phase Cell Cycle Checkpoints genetics   MeSH
• G2 Phase Cell Cycle Checkpoints genetics   MeSH
• Humans MeSH
• Human Embryonic Stem Cells cytology   metabolism   radiation effects   MeSH
• DNA Repair genetics   MeSH
• Cellular Reprogramming MeSH
• Cellular Senescence genetics   radiation effects   MeSH
• Gamma Rays MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• DNA MeSH
• H2AX protein, human MeSH Browser
• Histones MeSH
• TP53BP1 protein, human MeSH Browser