<p><span style="font-size:11pt">Background: Mesenchymal Stem Cells (MSCs) play a pivotal role in regenerative medicine due to their multipotency and immunomodulatory properties. However, during in vitro expansion, MSCs undergo senescence, characterized by a decline in proliferation, impairment of differentiation potential, and altered secretory profiles, which limits their therapeutic efficacy. This study aimed to identify novel molecular regulators and net-work-level interactions underlying MSC senescence through microarray analysis of the GSE7888 dataset, comparing early and senescent MSCs. </span></p> <p><span style="font-size:11pt">Methods: A total of 4597 Differentially Expressed Genes (DEGs) were identified be-tween early (passages 4-5) and senescent (passages 22-28) MSCs, with 2219 upregulated and 2379 downregulated. Key regulators such as CDKN1A (p21), CDK4, and CDK6 were implicated in cell cycle arrest and the progression of senescence. Pathway analysis highlighted the mTOR, FoxO, and p53 signaling pathways as key regulators of stress responses, metabolism, and aging. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis highlighted cellular processes, including protein transport and intracellular signaling, while protein-protein interaction networks identified high-connectivity nodes such as TP53, FOXO3, and MDM2. Senescent MSCs displayed phenotypic changes, including altered morphology and the emergence of the Senescence-Associated Secretory Phenotype (SASP), which impaired re-generative potential. </span></p> <p><span style="font-size:11pt">Results: The findings suggest that targeting the mTOR, FoxO, and p53 pathways could delay senescence and enhance MSC therapeutic potential. Interventions such as rapamycin and FoxO3 activators show promise in reversing senescence. </span></p> <p><span style="font-size:11pt">Conclusion: Future research should explore small molecules and gene-based therapies targeting senescence pathways to improve MSC-based regenerative strategies. These results provide a foundation for developing innovative approaches to optimize MSC ap-plications in clinical therapies.</span></p>