Cellular senescence is a fundamental hallmark of aging and represents a major barrier to the scalable and reproducible application of mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs). Senescent human umbilical cord mesenchymal stromal cells (hUCMSCs) exhibit impaired proliferative capacity, disrupted mitochondrial homeostasis, and altered secretory phenotypes, which may compromise the biological activity and therapeutic reliability of sEVs. Here, we investigated whether overexpression of human telomerase reverse transcriptase (hTERT) attenuates senescence-associated deterioration and stabilizes sEV functional properties. Late-passage hUCMSCs displayed canonical senescence features and mitochondrial dysfunction, all of which were markedly alleviated by hTERT expression. Functionally, sEVs derived from senescent cells exhibited impaired immunomodulatory activity, whereas sEVs from hTERT-expressing cells largely restored this function. Mechanistically, senescence was associated with altered sEV cargo, including enrichment of miR-217-5p, which contributed to diminished immunomodulatory potency, at least in part through modulation of SIRT1-associated inflammatory signaling in recipient macrophages. Proteomic profiling further showed that H11-sEVs retained a young-like protein cargo profile, particularly for proteins associated with immune and inflammatory regulation. In parallel, sEVs from all three groups retained primordial follicle-activating capacity, consistent with the broad retention of PI3K-Akt pathway-related proteins in their proteomic profiles and PI3K-Akt pathway activation in ovarian tissues. In aged female mice, H11-sEVs exhibited young-like ovarian protective activity. Collectively, these findings demonstrate that hTERT uncouples cellular senescence from sEV functional decline, supporting the development of potency-stabilized sEV sources for aging-related and regenerative applications and providing insight into the biological roles of senescent cell-derived sEVs.
