A Human Embryonic Stem Cell-derived Neural Stem Cell Senescence Model Triggered By Oxidative Stress.

Hui Pan, Li Bao, Meng Ji, Zhengbing Lyu, Nianmin Qi, Yuehong Wu
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Abstract

Introduction: Neural stem cells (NSCs) are vulnerable to oxidative stress, which triggers aging and subsequently leads to a reduced regenerative capacity of the central nervous system (CNS). Due to the challenges in acquiring aged human NSCs and the lack of an oxidative stressinduced aging model specifically designed for human NSCs, research related to the aging mechanisms and the screening of anti-aging drugs have been limited. Here, we aimed to establish an oxidative stress-induced senescence model of NSCs by using D-galactose (D-gal).

Methods: Human embryonic stem cells (hESC) were differentiated into hESC-NSCs using a type I collagen method. hESC-NSCs were characterized by flow cytometry combined with immunofluorescence. A senescence model of hESC-NSCs was established using D-gal and characterized by CCK-8 assay, neurosphere formation, crystal violet staining, DNA damage assay, SA-β-gal staining and ROS levels measurement. To further explore the profile of gene expression in D-gal-induced hESCNSCs senescence model, transcriptome sequencing was performed and analysed by bioinformatics method, following verified by qPCR.

Results: The hESC-derived NSCs senescence model demonstrated reduced proliferation and elevated β-galactosidase activity, accompanied by DNA damage and increased levels of reactive oxygen species. Furthermore, transcriptome analysis unveiled the potential central role of the MAPK signaling pathway in D-gal-induced senescence, which involves the key genes including DDIT3, ATF3, CEBPB, JUN, and CCND1.

Conclusion: We presented an oxidative stress-induced senescence model of hESC-NSCs and identified key pathways and genes related to D-gal-induced senescence. Our study might offer an alternative approach for investigating human NSC aging and provide valuable data for understanding the underlining mechanisms of oxidative stress-induced aging.

由氧化应激引发的人类胚胎干细胞衍生神经干细胞衰老模型
导言神经干细胞(NSCs)很容易受到氧化应激的影响,从而引发衰老,进而导致中枢神经系统(CNS)再生能力下降。由于获取衰老的人类 NSCs 存在困难,而且缺乏专门针对人类 NSCs 设计的氧化应激诱导衰老模型,因此有关衰老机制和抗衰老药物筛选的研究一直很有限。方法:采用Ⅰ型胶原法将人胚胎干细胞(hESC)分化为hESC-NSCs,用流式细胞术结合免疫荧光法对hESC-NSCs进行表征。利用D-gal建立了hESC-NSCs的衰老模型,并通过CCK-8检测、神经球形成、水晶紫染色、DNA损伤检测、SA-β-gal染色和ROS水平测量进行了表征。为了进一步探索 D-gal 诱导的 hESCNSCs 衰老模型的基因表达谱,研究人员进行了转录组测序,并通过生物信息学方法进行了分析,随后通过 qPCR 进行了验证:结果:源于 hESC 的 NSCs 衰老模型表现出增殖减少、β-半乳糖苷酶活性升高,同时伴有 DNA 损伤和活性氧水平升高。此外,转录组分析揭示了 MAPK 信号通路在 D-gal 诱导的衰老中的潜在核心作用,其中涉及的关键基因包括 DDIT3、ATF3、CEBPB、JUN 和 CCND1:我们提出了氧化应激诱导的 hESC-NSCs 衰老模型,并确定了与 D-gal 诱导衰老相关的关键通路和基因。我们的研究可能为研究人类 NSC 衰老提供了另一种方法,并为了解氧化应激诱导衰老的基本机制提供了有价值的数据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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