Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress - PubMed (original) (raw)

Human umbilical cord blood-derived mesenchymal stem cells undergo cellular senescence in response to oxidative stress

Eun Ko et al. Stem Cells Dev. 2012.

Abstract

Since human mesenchymal stem cells (MSCs) are therapeutically attractive for tissue regeneration and repair, we examined the physiological responses of human umbilical cord blood-derived MSCs (hUCB-MSCs) to genotoxic stress. We found that that sublethal doses of reactive oxygen species (ROS) and ionizing radiation cause DNA damage and reduce DNA synthesis and cell proliferation in hUCB-MSCs, resulting in cellular senescence. In contrast, these physiological changes were limited in human fibroblast and cancer cells. Our data show that reduced activities of antioxidant enzymes, which may occur due to low gene expression levels, cause hUCB-MSCs to undergo cellular senescence in response to oxidative stress and ionizing radiation. Resistance of hUCB-MSCs to oxidative stresses was restored by increasing the intracellular antioxidant activity in hUCB-MSCs via exogenous addition of antioxidants. Therefore, the proliferation and fate of hUCB-MSCs can be controlled by exposure to oxidative stresses.

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Figures

FIG. 1.

FIG. 1.

Proliferation of hUCB-MSCs is susceptible to genotoxic stresses. (A, B) hUCB-MSCs (MSC1, 2, 3, and 4), human fibroblast cells (MRC5 and HS68), and cancer cells (U2OS and HeLa) were treated with the indicated concentrations of hydrogen peroxide for 2 h as previously described [22]. The cells were washed twice with PBS and further incubated in fresh medium for 24 h, followed by immunostaining with the anti-Ki-67 (see also Supplementary Fig. S1) or anti-BrdU antibody for detection of cell proliferation or DNA synthesis, respectively. (C, D) Cells irradiated with γ-rays were grown for 24 h and immunostained as described previously. For the BrdU incorporation assay, cells were incubated for 30 min in the presence of 10 μM of BrdU prior to immunostaining as previously described [22]. DNA was visualized using DAPI staining. The proportion of Ki-67 or BrdU-positive cells was determined in each untreated or treated cell population; then the fold difference in proportion between treated and untreated cells is described on graphs as “Proliferation” or “DNA synthesis,” respectively. Graphs represent the averages of at least 3 independent experiments, detecting more than 100 cells each. The percentages (%) of untreated cells exhibiting Ki-67–positive staining were as follows: 90.5, U2OS; 87.5, HeLa; 73.9, HS68; 83.5, MRC5; 82.0, MSC1; 81.0, MSC2; 69.4, MSC3; and 58.8, MSC4. The percentages (%) of untreated cells exhibiting BrdU-positive staining were as follows: 33.3, U2OS; 37.3, HeLa; 36, HS68; 33.7, MRC5; 30.7, MSC1; 30.0, MSC2; 25.3, MSC3; and 28.5, MSC4. The P values were <0.0001 (A), <0.0001 (B), 0.0023 (C), and 0.0004 (D). MSCs, mesenchymal stem cells; hUCB-MSCs, human umbilical cord blood–derived MSCs; PBS, phosphate-buffered saline.

FIG. 2.

FIG. 2.

Oxidative stress causes more DNA breaks in hUCB-MSCs. (A, B) Cells incubated with the indicated concentration of hydrogen peroxide for 30 min (A) or 2 h (B) were analyzed in a comet assay to measure DNA damage. (C) The olive tail moment values of the 2-h samples were measured using the Komet 7.0 software and indicated as previously described [52,53]. The P value was 0.0124.

FIG. 3.

FIG. 3.

Recovery following DNA damage in hUCB-MSCs. (A, C) Cells treated with the indicated concentration of hydrogen peroxide for 30 min were washed with PBS and then incubated in fresh media for the indicated number of hours. Comet assays were then performed. Values of olive tail moment after treatment with 500 μM (B) or 50 μM (D) of hydrogen peroxide are presented. The P values were <0.0001 **(B)** and >0.05 (D).

FIG. 4.

FIG. 4.

Oxidative stress induces cellular senescence in hUCB-MSCs. (A) Cells incubated with 0 or 100 μM of hydrogen peroxide for 2 h were washed with PBS, and then incubated in fresh medium for 4 days. Cells were then stained with SA-β-Gal. (B) The percentage of cells stained blue (SA-β-Gal positive) is indicated. The data represent the average of 3 independent experiments, using over 150 cells each. (C) Cells incubated with 200 or 500 μM of hydrogen peroxide for 2 or 4 h were stained with trypan blue, and dead cells were scored using a hemocytometer. (D) Cells treated with 10 μM of phorbol-12-myristate-13-acetate for 4 h (see also Supplementary Fig. S5) were stained with trypan blue solution. The P values were <0.001 (B) and 0.0047 (D). SA-β-Gal, senescence-associated β-galactosidase.

FIG. 5.

FIG. 5.

hUCB-MSCs show low antioxidant activity. (A) Cells incubated with 20 μM of 2′,7′-dichlorodihydrofluorescein diacetate for 30 min were washed, and then incubated with fresh media containing the indicated concentration of hydrogen peroxide for 30 min. Intracellular ROS levels were detected using the FACS Calibur instrument (BD Bioscience). Mean fluorescence intensity of DCF was used as measure of ROS, and values were normalized with the basal intensity of each cell line. (B) Total antioxidant activity in each cell lysate was measured using Trolox E as described in the Materials and Methods section. Values per 1 μg of cell lysate are shown. (C) Activities of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were measured as described in the Materials and Methods section. Enzyme activity per 1 μg of cell lysate is indicated. (D) 40 μg of total protein from each cell line was immunoblotted with the appropriate antibodies (see also Supplementary Fig. S6A). Relative band intensities normalized to the β-actin band were calculated using Multi Gauge 3.0 software (Fujifilm). SOD1, superoxide dismutase 1; SOD2, superoxide dismutase 2; GPx1, glutathione peroxidase 1. (E) 1 μg of mRNA from each cell line was reverse transcribed using specific primers (Materials and Methods). Relative band intensities from gel electrophoresis following RT-PCR (Supplementary Fig. S6B) were measured using Multi Gauge 3.0 software. Quantitative data for each RT-PCR product were normalized against the GAPDH level. The P values were 0.036 (A); 0.017 (B); 0.0081, catalase activity in (C); 0.0076, SOD activity in (C); and <0.0001, GPx activity in (C). DCF, 2′,7′-dichlorodihydrofluorescein; ROS, reactive oxygen species.

FIG. 6.

FIG. 6.

Exogenous addition of antioxidant prevents hUCB-MSCs from undergoing cellular senescence and cell death due to oxidative stress. (A) Cells incubated with 200 U/mL of PEG-catalase (PEG-Cat) for 24 h were further incubated for 2 h in fresh medium containing the indicated concentrations of hydrogen peroxide. The cells were then transferred to fresh medium, grown for 24 h, and immunostained using anti-Ki-67 antibodies (see also Supplementary Fig. S7A, B). (B) Cells pretreated with 200 U/mL of PEG-catalase were incubated in fresh medium for 2 h with hydrogen peroxide. Four days after hydrogen peroxide treatment, the cells were stained using SA-β-Gal. The percentage of SA-β-Gal–positive cells is indicated in each micrograph. (C) PEG-catalase (200 U/mL)–pretreated cells were incubated with 500 μM of hydrogen peroxide for 2 or 4 h. Cells were observed under a phase-contrast microscope (Supplementary Fig. S7C), and harvested cells were stained with trypan blue solution and counted. (D) hUCB-MSC lines, MSC1 and MSC2, were incubated with 1 mM of _N_-acetyl cysteine (NAC) for 6 h, and then incubated for 2 h in fresh medium containing the indicated concentrations of hydrogen peroxide. Cells were viewed under a phase-contrast microscope, and dead cells were counted using trypan blue staining. The graph shows the results from 3 independent experiments. The P values were <0.0001 (C) and 0.011 (D). PEG-catalase, polyethylene glycol–conjugated catalase.

FIG. 7.

FIG. 7.

Increased antioxidant activity inhibits hUCB-MSCs from undergoing cellular senescence following ionizing radiation. (A) MSC1 hUCB-MSCs, pretreated with 200 U/mL of PEG-catalase for 24 h, were irradiated with γ-rays. One day after irradiation, cells were immunostained using the anti-Ki-67 antibody (upper panel). DAPI staining was used to visualize nuclei. The relative proportion of Ki-67–stained cells is shown in the plot (lower panel). The results of 3 independent experiments were averaged. P value was 0.037. (B) Two or 4 days after irradiation, MSC1 cells were stained using SA-β-Gal and viewed under a phase-contrast microscope (upper panel). The graph represents the percentage of SA-β-Gal–stained cells at 4 days after irradiation (lower panel). IR, γ-irradiation.

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