College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China.
Taixing Animal Husbandry and Veterinary Center, Taizhou, Jiangsu Province, China.
Cell Cycle. 2023 Jul;22(13):1597-1613. doi: 10.1080/15384101.2023.2222521. Epub 2023 Jun 20.
Epigenetic status of fetal fibroblasts (FFs) is one of the crucial factors accounted for the success of somatic cell nuclear transfer and gene editing, which might inevitably be affected by passaging. But few systematic studies have been performed on the epigenetic status of passaged aging cells. Therefore, FFs from large white pig were passaged to the 5, 10, and 15 (F5, F10, and F15) passages in the present study to investigate the potential alteration of epigenetic status. Results indicated the senescence of FFs occurs with the passaging, as assessed by the weakened growth rate, increased β-gal expression, and so on. For the epigenetic status of FFs, the higher level both of DNA methylation and H3K4me1, H3K4me2, H3K4me3 was observed at F10, but the lowest level was observed at F15. However, the fluorescence intensity of m6A was significantly higher in F15, but lower ( < 0.05) in F10, and the related mRNA expression in F15 was significantly higher than F5. Further, RNA-Seq indicated a considerable difference in the expression pattern of F5, F10, and F15 FFs. Among differentially expressed genes, not only the genes involved in cell senescence were changed, but also the upregulated expression of Dnmt1, Dnmt3b, Tet1 and dysregulated expression of histone methyltransferases-related genes were detected in F10 FFs. In addition, most genes related to m6A such as , , and were significantly different in F5, F10, and F15 FFs. In conclusion, the epigenetic status of FFs was affected by being passaged from F5 to F15.
成纤维细胞（FFs）的胚胎状态是影响体细胞核移植和基因编辑成功的关键因素之一，而其可能会受到传代的影响。但是，对于传代老化细胞的胚胎状态，很少有系统的研究。因此，本研究对大白猪的 FF 进行了传代，传至第 5、10 和 15 代（F5、F10 和 F15），以研究其胚胎状态的潜在变化。结果表明，FF 的衰老与传代有关，如生长速度减弱、β-gal 表达增加等。对于 FF 的胚胎状态，在 F10 时观察到 DNA 甲基化和 H3K4me1、H3K4me2、H3K4me3 的水平较高，而在 F15 时则较低。然而，F15 的 m6A 荧光强度明显较高，而 F10 的则明显较低（<0.05），F15 的相关 mRNA 表达明显高于 F5。此外，RNA-Seq 表明 F5、F10 和 F15 FF 的表达模式存在显著差异。在差异表达的基因中，不仅参与细胞衰老的基因发生了变化，而且在 F10 FF 中还检测到 Dnmt1、Dnmt3b、Tet1 的上调表达和组蛋白甲基转移酶相关基因的失调表达。此外，大多数与 m6A 相关的基因，如、和 ，在 F5、F10 和 F15 FF 中差异显著。综上所述，FF 从 F5 传代至 F15 会影响其胚胎状态。
