Allegrucci Cinzia, Wu Yue-Zhong, Thurston Alexandra, Denning Chris N, Priddle Helen, Mummery Christine L, Ward-van Oostwaard Dorien, Andrews Peter W, Stojkovic Miodrag, Smith Nigel, Parkin Tony, Jones Mark Edmondson, Warren Graham, Yu Li, Brena Romulo Martin, Plass Christoph, Young Lorraine E
Division of Obstetrics and Gynaecology and Wolfson Centre for Stem Cells, Tissue Engineering and Modelling (STEM), University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
Hum Mol Genet. 2007 May 15;16(10):1253-68. doi: 10.1093/hmg/ddm074. Epub 2007 Apr 4.
Widespread provision of human embryonic stem cells (hESCs) for therapeutic use, drug screening and disease modelling will require cell lines sustainable over long periods in culture. Since the short-term, in vitro culture of mammalian embryos can result in DNA methylation changes, the epigenetic stability of hESCs warrants investigation. Existing hESC lines have been derived and cultured under diverse conditions, providing the potential for programming differential changes into the epigenome that may result in inter-line variability over and above that inherited from the embryo. By examining the DNA methylation profiles of > 2000 genomic loci by Restriction Landmark Genome Scanning, we identified substantial inter-line epigenetic distance between six independently derived hESC lines. Lines were found to inherit further epigenetic changes over time in culture, with most changes arising in the earliest stages post-derivation. The loci affected varied between lines. The majority of culture-induced changes (82.3-87.5%) were stably inherited both within the undifferentiated cells and post-differentiation. Adapting a line to a serum-free culture system resulted in additional epigenetic instability. Overall 80.5% of the unstable loci uncovered in hESCs have been associated previously with an adult tumour phenotype. Our study shows that current methods of hESC propagation can rapidly programme stable and unpredictable epigenetic changes in the stem cell genome. This highlights the need for (i) novel screening strategies to determine the experimental utility and biosafety of hESCs and (ii) optimization and standardization of procedures for the derivation and culture of hESC lines that minimize culture-induced instability.
广泛提供用于治疗、药物筛选和疾病建模的人类胚胎干细胞（hESCs）需要能够在长期培养中保持稳定的细胞系。由于哺乳动物胚胎的短期体外培养会导致DNA甲基化变化，因此hESCs的表观遗传稳定性值得研究。现有的hESC系是在不同条件下获得和培养的，这有可能在表观基因组中编程产生差异变化，从而导致细胞系间的变异性超出从胚胎继承的范围。通过限制性地标基因组扫描检查2000多个基因组位点的DNA甲基化谱，我们在六个独立获得的hESC系之间发现了显著的细胞系间表观遗传距离。发现细胞系在培养过程中会随着时间进一步继承表观遗传变化，大多数变化发生在获得后的早期阶段。不同细胞系受影响的位点各不相同。大多数培养诱导的变化（82.3 - 87.5%）在未分化细胞内和分化后都能稳定遗传。使一个细胞系适应无血清培养系统会导致额外的表观遗传不稳定性。总体而言，在hESCs中发现的80.5%的不稳定位点先前已与成人肿瘤表型相关。我们的研究表明，目前hESC的培养方法可以在干细胞基因组中快速编程产生稳定且不可预测的表观遗传变化。这突出了需要（i）新的筛选策略来确定hESCs的实验效用和生物安全性，以及（ii）优化和标准化hESC系的获得和培养程序，以尽量减少培养诱导的不稳定性。
