Abstract: Assisted reproductive technology (ART) has gained increasing significance amidst declining birth rates. However, its efficacy is challenged by aging, particularly the decline in oocyte quality observed in individuals in their mid-30s. Our research focuses on the molecular mechanisms that underlie the age-related deterioration of stem cell function, aiming to pave the way for improved outcomes in ART for older individuals. Mouse embryonic stem cells (ESCs) were isolated from parthenogenetically developed blastocysts. Induced pluripotent stem cells (iPSCs) were isolated from fibroblast of isogenic mice at three age groups: young (6–8 weeks), middle (6 months) and aged (12–30 months). RNA sequencing, radiation-induced genome stability assays, and telomere length quantification analyses were performed. Stem cells derived from all ages demonstrated robust self-renewal capacity and maintained pluripotency, as validated by immunohistochemistry and quantitative PCR. Hierarchical clustering of RNA-seq data revealed that aged iPSCs were transcriptomically distinct from young iPSCs and ESCs. Gene ontology analysis highlighted significant downregulation of genes involved in chromosome segregation, chiasma assembly, nuclear division and cell cycle processes in aged iPSCs. Intriguingly, transcriptomic profiling revealed upregulation of zygotic genome activation (ZGA) genes in aged iPSCs. Radiation-induced genome stability assays demonstrated a marked increase in γH2AX foci—an indicator of DNA damage—in iPSCs with aging, reflecting elevated genomic instability. Interestingly, knockdown of ZGA genes via small hairpin RNA modestly reduced γH2AX foci in aged iPSCs. The average number of foci decreased from 5.4 (control) to 4.8 (knockdown shRNA group) in young iPSCs. In aged iPSCs, the number decreased from 7.5 (control) to 6.5 (knockdown shRNA group). Notably, telomere length analysis did not reveal any significant differences between young and aged groups. Our findings suggest the presence of a compensatory ZGA gene cluster in aged stem cells that may mitigate the effects of aging. These insights pave the way for innovative strategies to enhance the developmental potential of aged embryos, potentially improving ART outcomes for older individuals.
