The replicative senescent mesenchymal stem / stromal cells defect in DNA damage response and anti-oxidative capacity

Abstract

Replicative senescence and potential malignant transformation are great limitations in the clinical application of bone marrow-derived mesenchymal stem / stromal cells (MSCs). An abnormal DNA damage response may result in genomic instability, which is an integral component of aging and tumorigenesis. However, the effect of aging on the DNA damage response in MSCs is currently unknown. In the present study, we evaluated the DNA damage response induced by oxidative stress and DNA double-strand breaks in human bone marrow-derived MSCs. After long-term cell culture, replicative senescent MSCs (sMSCs) were characterized by a poor proliferation rate, high senescence-associated β-galactosidase activity, and enhanced expression of P53 and P16. Features of the DNA damage response in these sMSCs were then compared with those from early-passage MSCs. The sMSCs were more sensitive to hydrogen peroxide and bleomycin treatment with respect to cell viability and apoptosis induction. Combined with the comet assay, γH2AX foci characterization and reactive oxygen species detection were used to demonstrate that the antioxidant and DNA repair ability of sMSCs are attenuated. This result could be explained, at least in part, by the downregulation of anti-oxidation and DNA repair genes, including Cu/Zn-SOD, GPX, CAT, OGG1, XRCC1, Ku70, BRCA2 and XRCC4. In conclusion, MSCs aging is associated with a reduction in the DNA repair and anti-oxidative capacity.

Keywords: DNA damage response; DNA double-strand breaks; Replicative senescence; mesenchymal stem cells; oxidative stress.

Figure 1

Establishment of a replicative senescent MSCs model. A. morphology of human bone marrow derived MSCs during long term in vitro culture. B. Growth curve of cultured MSCs. C. FCM characterization of MSCs. D. Osteogenic and Adipogenic differentiation of MSCs was confirmed by staining with Alizarin red and Oil Red O, respectively. E. SA-β-gal staining of yMSCs, counter stained with hematoxylin. F. Real-time RT-PCR showing the expression of p16 and p53 in yMSCs and sMSCs. * indicated that p < 0.05, ** indicated that p < 0.01.

Figure 2

sMSCs are more sensitive to H₂O₂ and BLM as compared with yMSCs. A and B. CCK-8 analysis of yMSCs and sMSCs after treatment of increasing concentration of H₂O₂ for 4h and cultured for 72h, or increasing concentration of BLM for 24h and cultured for 48h. C and D. Apoptosis detection in yMSCs and sMSCs. The cells were treated with 600μM of H₂O₂ for 4h and cultured for 24h (C) or treated with 30μg/ml BLM for 24h and cultured for 24h (D), and were then stained with Annexin V (green) and P I(red). E. Quantification of C and D. Data are presented as mean value±SD of three independent experiments. ** indicated that p < 0.01.

Figure 3

The antioxidant ability is decreased in the sMSCs. A. The intracellular level of ROS was measured by the fluorescent dye DCFH-DA in MSCs without treatment(control), after 2h treatment with 100 μM, 300 μM or 600μM H₂O₂. B. The quantitation result of A. C. Real time-PCR analysis of the basal expression level of anti-oxdative Enzymes genes Cu/Zn-SOD、Mn-SOD, GPX and CAT in yMSCs and sMSCs. Data are presented as mean values±SD of three independent experiments. * indicated that p < 0.05, ** indicated that p < 0.01.

Figure 4

The expressions of DNA repair genes are downregulated in sMSCs. A to C. Real time-PCR analysis of BER genes including OGG1, XRCC1 and APE1 in cells at 6, 12 and 24 hours after 300 μM H₂O₂ treated for 2 hours. D, E. Real time-PCR analysis of DSB repair genes including BRCA1, BRCA2, Rad51, Ku70 and XRCC4 in cells at 0.5, 2, 6, 12 and 24 hours after 50μg/ml BLM treated for 2 hours. Data are presented as mean value±SD of three independent experiments. * indicated that p < 0.05, ** indicated that p < 0.01.

Figure 5

The DNA repair ability was attenuated in sMSCs. A. Alkaline comet assay of MSCs after 25 μM of H₂O₂treatmented. B. Quantification of OTM scores of A, 100 randomly chosen comets were analyzed. C. Alkaline comet assay of MSCs after 100 μM of H₂O₂ treatment. D. Quantification of OTM scores of C, 100 randomly chosen comets were analyzed. E. Neutral comet assay of MSCs after 30 μg/ml BLM treatment. F. Quantification of OTM scores of E, 100 randomly chosen comets were analyzed. G. Immunofluorescence microscopy of γH2AX foci in MSCs after 30 μg/ml BLM treatment. H. Quantification of γH2AX foci number in G, at least 100 randomly chosen nucleuses were analyzed and plotted. Data are presented as mean value±SD of three independent experiments. * indicated that p < 0.05, ** indicated that p < 0.01.