α-酮戊二酸抑制人原始态多能干细胞的长期稳态维持

α-Ketoglutarate Impairs the Long-Term Maintenance of Naïve Human Pluripotent Stem Cells

该文探讨了4CL条件下人原始态多能干细胞(naïve human pluripotent stem cells,nPSCs)长期传代过程中自我更新能力和多能性基因表达水平的下降及其机制,并测试了α-酮戊二酸(alphaketoglutarate,αKG)对nPSCs稳态维持的影响。通过细胞生长曲线、RT-qPCR实验分别检测连续传代过程中以及αKG处理后细胞增殖能力和多能性基因表达水平的变化;采用转录组测序分析长期传代和αKG处理后的差异表达基因,并进行功能分析和motif预测。结果显示,4CL nPSCs连续传代过程中细胞增殖能力和多能性基因表达水平均逐渐降低,同时伴随分化相关基因表达变化;转录组分析发现4CL nPSCs的自我更新能力下降可能与p53通路激活有关,而多能性基因表达水平下降则可能与ETS、FOX家族转录因子有关。添加αKG处理,短期并未显著改变4CL nPSCs多能性基因的表达,反而对自我更新产生一定的抑制作用;长期虽然在一定程度上提高了4CL nPSCs的自我更新能力,但却进一步降低了多能性基因的表达水平。总之,4CL nPSCs长期传代后稳定性下降,添加αKG后,其稳定性进一步降低。

This study aimed to investigate the decline in self-renewal capacity and pluripotency gene expression in nPSCs(naïve human pluripotent stem cells)during long-term passaging under 4CL conditions and to explore the underlying mechanisms.Additionally,the effect ofαKG(alpha-ketoglutarate)on the steady-state maintenance of nPSCs was examined.Cell proliferation and pluripotency gene expression levels separately were assessed through cell growth curves and RT-qPCR during continuous passaging and afterαKG treatment.Transcriptome sequencing was used to analyze differentially expressed genes after long-term passaging andαKG treatment,followed by functional analysis and motif prediction.Results indicated that cell proliferation and pluripotency gene expression gradually decreased during the continuous passaging of 4CL nPSCs,and were accompanied by changes in the expression of differentiation-related genes.Transcriptome analysis suggested that the decline in self-renewal might be associated with the activation of the p53 pathway,while the decrease in pluripotency gene expression could be linked to ETS and FOX family transcription factors.Short-termαKG treatment inhibited self-renewal without altering pluripotency gene expression,whereas long-term treatment improved self-renewal to some extent but further reduced pluripotency gene expression.In summary,the stability of 4CL nPSCs declines after long-term passaging,and this stability is further compromised byαKG treatment.