Abstract
BACKGROUND: Mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but their clinical utility is limited by replicative senescence. Strategies that reverse aging while maintaining MSC identity are urgently needed. METHODS: We developed a non-integrating, temperature-sensitive Sendai virus (SeV)-mediated rejuvenation protocol transiently expressing hTERT, BMI1, and SV40T in human MSCs. Following SeV removal, we evaluated proliferation, telomere length, karyotype stability, transcriptomic reset, producing heterogeneity, and differentiation potential. RESULTS: Rejuvenated MSCs (rej-MSCs) demonstrated extended proliferation beyond 100 days, telomere elongation, and normal karyotypes after SeV clearance. Transcriptomic profiling showed a reset of senescence-associated programs while retaining mesenchymal identity. Functional analyses revealed clone-specific heterogeneity, including HGF-driven angiogenic activity. Multilineage differentiation capacity was preserved across rej-MSCs. CONCLUSIONS: This transient, non-integrating rejuvenation strategy establishes an operational definition of rej-MSCs and provides a transcriptionally diverse and scalable platform for MSC manufacturing and precision therapy design.