L level of antioxidants in medium is enough or not. Interestingly, we’ve lately discovered a biphasic effect of antioxidants on genomic stability of stem cells9. We found that the supplement of low dosages of antioxidant cocktails probably contribute to the lower DNA harm and also the DPP-4 Inhibitor Formulation improvement of genomic stability of stem cells, conversely, higher dosages of antioxidants improve the threat of chromosomal Histamine Receptor Modulator Formulation abnormalities of stem cells by interfering with the endogenous DNA repair pathways. Herein, we examined no matter whether the supplement of low dosages of antioxidants in culture medium could improve the high quality and genomic stability of induced pluripotent stem (iPS) cells during long-term ex vivo expansion.Final results Low dose antioxidants did not influence the growth and “stemness” of iPS cells. We effectively maintained the iPS cell lines for two months by often passage. The shape and development of iPS cell colonies were not certainly changed by adding either proprietary antioxidant supplement from Sigma-Aldrich (AOS) or homemade antioxidant cocktail (AOH) at relative low concentrations in culture medium for two months of follow-up. Immunostaining showed that all of those iPS cell colonies clearly expressed Oct3/4, Nanog, SSEA-4, and ALPSCIENTIFIC REPORTS | four : 3779 | DOI: 10.1038/srep03779nature/scientificreportsFigure 1 | Stemness of iPS cells after two months of culture. The expression of stem cell markers Oct3/4, Nanog, SSEA-4, and ALP have been detected by staining, and representative pictures of expressions in 201B7 (A) and 253G1 (B) iPS cell lines have been shown. Western blot evaluation around the expressions of Nanog and Oct3/4 in 201B7 (C) and 253G1 (D) iPS cell lines was also accomplished, and representative pictures that cropped from full-length blots (Supplementary Figure 1) was shown. Abbreviations: AOS, proprietary antioxidant supplement from Sigma-Aldrich; AOH, Homemade antioxidant cocktail.soon after two months (Figure 1A and B), indicating that all culture circumstances maintained “stemness” of iPS cells quite well. Western blot analysis also showed that the expressions of Nanog and Oct3/ 4 at comparable higher levels in all iPS cells under various culture circumstances (Figure 1C and D), though the expressions had been not cautiously quantified. Low dose antioxidants decreased the intracellular ROS levels in iPS cells. We initially measured ROS level by detecting the fluorescence intensity under microscope (Figure 2A). When compared with the handle, the addition of proprietary antioxidant supplement from Sigma-Aldrich or homemade antioxidant cocktail at relative low concentrations in culture medium certainly decreased the levels of intracellular ROS within the iPS cells (upper photos in Figure 2A). Semiquantitative analysis showed that the relative fluorescence intensity of intracellular ROS had been drastically reduced within the iPS cells cultured with all the addition of antioxidants in medium than that of the control (reduced bar graphs in Figure 2A). To additional quantitative measure the ROS levels, we measured the fluorescence intensity in iPS cells by flow cytometry (Figure 2B). Again, the addition of antioxidants in medium showed to substantially lower the ROS levels within the iPS cells, although the reduce of ROS by antioxidants was not clearly shown within a dose-dependent manner. Low dose antioxidants did not market DNA damage or inhibit DNA repair in iPS cells. We evaluated the DNA harm by counting the formation of 53BP1 foci within the nuclei of iPS cells just after two months culture together with the.
