While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

中文翻译：

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延长人类胚胎干细胞培养过程中肿瘤抑制基因甲基化模式的变化

虽然对胚胎干细胞的研究一直在积极进行，但对扩展培养系统中人类胚胎干细胞 (hESC) 的表观遗传机制知之甚少。在这里，我们调查了 24 个肿瘤抑制基因的 CpG 岛 (CGI) 甲基化模式是否可以在扩展的 hESC 培养过程中保持。总共分析了 10 个 hESC 系。对于每个细胞系，从细胞培养物的早期和晚期传代中提取基因组 DNA。使用甲基化特异性多重连接依赖性探针扩增 (MS-MLPA)、焦磷酸测序和实时聚合酶链反应 (PCR) 分析了 24 个肿瘤抑制基因的 CGI 甲基化水平。CASP8、FHIT和CHFR的不同CGI甲基化模式在一些 hESC 系的早期和晚期传代之间鉴定了基因。与早期传代相比，CASP8 的CGI 甲基化水平在 CHA-36、CHA-40 和 CHA-42 细胞系中在传代后期显着增加。FHIT基因的 CGI 甲基化在 CHA-15、CHA-31、CHA-32 和 iPS (FS)-1 细胞系中晚期传代高于早期传代，但在 CHA-20 和 H1 晚期传代时降低细胞系。CHFR检测到不同的 CGI 甲基化模式基因仅在 iPS (FS)-1 中存在，并且在传代后期水平显着增加。因此，我们的研究结果表明，在长期的 ESC 培养过程中，CGI 甲基化模式可能会发生改变，检查这些表观遗传变化对于评估干细胞的维持、分化和临床使用很重要。