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A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells.

Alves H, Munoz-Najar U, De Wit J, Renard AJ, Hoeijmakers JH, Sedivy JM, Van Blitterswijk C, De Boer J - J. Cell. Mol. Med. (2010)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. hugoandrealves@gmail.com

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Activation of the DNA damage signalling pathway and efficiency of NER. Samples from different RPD were analysed by Western blotting for p16 and p21 protein expression and p53 and BRCA1 protein phosphorilation (A). β-actin served as loading control. NER efficiency was determined at several RPD after in vitro expansion using the unscheduled DNA synthesis assays. Error bars represent standard deviation. Statistical analysis was performed using one-way anova and Tukey post-test with a significance level of 0.05. **P < 0.01 and ***P < 0.001.
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fig03: Activation of the DNA damage signalling pathway and efficiency of NER. Samples from different RPD were analysed by Western blotting for p16 and p21 protein expression and p53 and BRCA1 protein phosphorilation (A). β-actin served as loading control. NER efficiency was determined at several RPD after in vitro expansion using the unscheduled DNA synthesis assays. Error bars represent standard deviation. Statistical analysis was performed using one-way anova and Tukey post-test with a significance level of 0.05. **P < 0.01 and ***P < 0.001.

Mentions: Next, we performed Western blot analysis on hMSCs at different RPDs and analysed the abundance of proteins involved in the DNA damage response pathway, such as phospho-p53 (Ser15), p16 (c-20) and p21Waf1/Cip1 (Fig. 3A). In accordance with the previous results, we observed an activation of the DNA damage response pathway with time of culture. There was a particularly strong increase from RPD 10 to RPD 17. We suggested that DNA damage accumulation is due to impaired DNA repair mechanisms in late passage cells. We analysed the capacity of NER because it is involved in the repair of many types of DNA damage including the ones induced by reactive oxygen species associated with premature ageing phenotypes. To this end, hMSC from different RPD were seeded onto coverslips and irradiated with UV after which we measured the incorporation of 3H-thymidine. With increased PD, there was no significant change in the NER capacity (Fig. 3B).


A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells.

Alves H, Munoz-Najar U, De Wit J, Renard AJ, Hoeijmakers JH, Sedivy JM, Van Blitterswijk C, De Boer J - J. Cell. Mol. Med. (2010)

Activation of the DNA damage signalling pathway and efficiency of NER. Samples from different RPD were analysed by Western blotting for p16 and p21 protein expression and p53 and BRCA1 protein phosphorilation (A). β-actin served as loading control. NER efficiency was determined at several RPD after in vitro expansion using the unscheduled DNA synthesis assays. Error bars represent standard deviation. Statistical analysis was performed using one-way anova and Tukey post-test with a significance level of 0.05. **P < 0.01 and ***P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3539748&req=5

fig03: Activation of the DNA damage signalling pathway and efficiency of NER. Samples from different RPD were analysed by Western blotting for p16 and p21 protein expression and p53 and BRCA1 protein phosphorilation (A). β-actin served as loading control. NER efficiency was determined at several RPD after in vitro expansion using the unscheduled DNA synthesis assays. Error bars represent standard deviation. Statistical analysis was performed using one-way anova and Tukey post-test with a significance level of 0.05. **P < 0.01 and ***P < 0.001.
Mentions: Next, we performed Western blot analysis on hMSCs at different RPDs and analysed the abundance of proteins involved in the DNA damage response pathway, such as phospho-p53 (Ser15), p16 (c-20) and p21Waf1/Cip1 (Fig. 3A). In accordance with the previous results, we observed an activation of the DNA damage response pathway with time of culture. There was a particularly strong increase from RPD 10 to RPD 17. We suggested that DNA damage accumulation is due to impaired DNA repair mechanisms in late passage cells. We analysed the capacity of NER because it is involved in the repair of many types of DNA damage including the ones induced by reactive oxygen species associated with premature ageing phenotypes. To this end, hMSC from different RPD were seeded onto coverslips and irradiated with UV after which we measured the incorporation of 3H-thymidine. With increased PD, there was no significant change in the NER capacity (Fig. 3B).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. hugoandrealves@gmail.com

Show MeSH
Related in: MedlinePlus