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

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 has 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 (hESCs) 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 the D-gal-induced hESC-NSCs senescence model, transcriptome sequencing was performed and analysed by bioinformatics method, followed by verification using 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, involving 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 to investigating human NSCs aging and provide valuable data for understanding the underlying mechanisms of oxidative stress-induced aging.