Abstract
Somatic cells change continuously during culture expansion—long-term culture evokes increasing cell size, declining differentiation potential, and ultimate cell cycle arrest upon senescence. These changes are of particular relevance for cellular therapy which necessitates standardized products and reliable quality control. Recently, replicative senescence has been shown to be associated with highly reproducible epigenetic modifications. Here, we describe a simple method to track the state of senescence in mesenchymal stromal cells (MSCs) or fibroblasts by monitoring continuous DNA methylation (DNAm) changes at specific sites in the genome. Six CpG sites have been identified which reveal either linear hypermethylation or hypomethylation with respect to the number of cumulative population doublings (cPDs). Conversely, the DNAm level at these CpG sites can be analyzed—for example, by pyrosequencing of bisulfite-converted DNA—and then used for linear regression models to predict cPDs. Our method provides an epigenetic biomarker to determine the state of senescence in cell preparations.
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Acknowledgments
We thank Anne Schellenberg for critical reading of the manuscript and Qiong Lin for contribution of the multivariate model. RWTH Aachen Medical School has applied for a patent application for the above mentioned method. This work was supported by the excellence initiative of the German federal and state governments within the START Program of the Faculty of Medicine, RWTH Aachen (W.W.), by the Stem Cell Network North Rhine Westphalia (W.W.) and by the Else-Kröner Fresenius Stiftung (W.W. and C.K.).
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Koch, C.M., Wagner, W. (2013). Epigenetic Biomarker to Determine Replicative Senescence of Cultured Cells. In: Tollefsbol, T. (eds) Biological Aging. Methods in Molecular Biology, vol 1048. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-556-9_20
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DOI: https://doi.org/10.1007/978-1-62703-556-9_20
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