Koch Carmen M, Wagner Wolfgang
Stem Cell Biology and Cellular Engineering, Helmholtz-Institute for Biomedical Engineering; RWTH Aachen University Medical School, Aachen, Germany.
Methods Mol Biol. 2013;1048:309-21. doi: 10.1007/978-1-62703-556-9_20.
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.
在培养扩增过程中，体细胞会持续发生变化——长期培养会导致细胞体积增大、分化潜能下降，并在衰老时最终导致细胞周期停滞。这些变化对于细胞治疗尤为重要，因为细胞治疗需要标准化的产品和可靠的质量控制。最近，已证明复制性衰老与高度可重复的表观遗传修饰有关。在此，我们描述了一种简单的方法，通过监测基因组中特定位点的连续DNA甲基化（DNAm）变化来追踪间充质基质细胞（MSC）或成纤维细胞的衰老状态。已鉴定出六个CpG位点，它们相对于累积群体倍增数（cPDs）显示出线性高甲基化或低甲基化。相反，这些CpG位点的DNAm水平可以通过例如对亚硫酸氢盐转化的DNA进行焦磷酸测序来分析，然后用于线性回归模型以预测cPDs。我们的方法提供了一种表观遗传生物标志物，用于确定细胞制剂中的衰老状态。
