Compartment-specific eccDNA patterns reveal senescence associated biomarkers in hUC-MSCs

The therapeutic potential of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) is significantly constrained by replicative senescence during in vitro expansion. To investigate this phenomenon, we established a long-term passaging model of hUC-MSCs and validated senescence-associated phenotypes. Through integrated transcriptomic and eccDNA profiling, we systematically analyzed three compartments—cell pellets (CP), cell culture media (CM), and extracellular vesicles (EVs)—at sequential timepoints (days 5, 22 and 43, post-seeding). Intriguingly, while eccDNA abundance in CP remained stable during senescence progression, CM and EVs exhibited higher eccDNA loads, respectively, compared to CP. Moreover, CM- and EVs-derived eccDNAs shared conserved size distribution patterns distinct from CP-associated eccDNAs. Furthermore, the compartment-specific eccDNA dynamics over time coincided with activation of growth-regulatory gene expression. We identified recurrent eccDNA species across all compartments, harboring genes linked to senescence-associated processes. Our study uncovers compartment-specific eccDNA dynamics during hUC-MSC aging and proposes their potential as biomarkers for senescence monitoring. These findings provide a foundation for developing strategies to mitigate senescence-related limitations in hUC-MSC clinical applications.