A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells.
Bottom Line: Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications.Unfortunately, this leads to a significant decrease in their stemness.To identify the mechanism behind loss of multi-potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals.
Affiliation: Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. email@example.comShow MeSH
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Mentions: First, we established a dose-response curve of hydrogen peroxide on proliferation of hMSCs. Up to 50 μM H2O2, the differences observed were not statistically significant (Fig. 4A). For paraquat, a concentration of 5 μM and 10 μM was used based on our previous experiments (data not shown). Based on this, we choose two concentrations of H2O2 (30 and 50 μM) and two of paraquat (5 and 10 μM) to treat hMSCs, where after 6 days, both DNA damage accumulation and ALP induction was assessed and compared to the control situation. Under these conditions, DNA damage accumulation occurred evidenced by the increase in 53BP1 positive cells [33.33 ± 4.9% in the control situation to 52.83 ± 6% in 30 μM H2O2 treated hMSCs and 51.8 ± 4.7 in 5 μM paraquat treated cells, (Fig. 4B)] and the number of 53BP1 foci per cell which increased from 0.50 ± 0.05 to 1.68 ± 0.19 (H2O2, 30 μM) or 0.77 ± 0.06 in (paraquat, 5 μM) in treated cells (Fig. 4C). When a stronger concentration of DNA damage inducer was used, both the percentage of damaged cells (53BP1 positive, Fig. 4B) and average foci per cell (Fig. 4C) increased even further. When a DNA-damage inducer was used, an increase in the β-galactosidase activity was also observed (Fig. S3). Exposure of hMSCs for 6 days to DNA damage inducers did not have an effect on ALP activity in BM (except for cells treated with 50 μM H2O2), but it did significantly inhibit dexamethasone-induced ALP expression in all the conditions tested (Fig. 4D). Mineralization was also significantly repressed by the usage of any of the inducers (Fig. 4E).
Affiliation: Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. firstname.lastname@example.org