Characterization and validation of long-term cultured TERT-immortalized human Wharton’s jelly–derived mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) have demonstrated efficacy and feasibility in numerous preclinical and clinical studies. However, their application is limited at early passages owing to replicative senescence. This study investigated whether MSCs immortalized via human telomerase reverse transcriptase (TERT) overexpression (TERT-MSCs) maintain their characteristics and efficacy after extended passaging. Human Wharton’s jelly-derived MSCs at passage (P) 6 were immortalized by retroviral transfection with human TERT. TERT-MSCs at mid (P13) and late (P30) passages were analyzed. Their morphology, differentiation potential, and surface marker expression were preserved throughout extended passaging. Single-cell RNA sequencing reconfirmed the characteristics of TERT-MSCs and compared their gene expression profiles with those of P8 parental (control) cells. As passage number increased, control MSCs exhibited gradual declines in TERT expression, telomere length, and cytoprotective effects, whereas both P13 and P30 TERT-MSCs retained these properties. Expression levels of paracrine-related genes and enriched Gene Ontology terms in extended-passage TERT-MSCs were comparable to those of control MSCs. These findings suggest that TERT-MSCs maintain the key characteristics and therapeutic potential of early-passage MSCs through extended culture, offering a promising approach to overcome the limitations of early-passage MSCs in clinical applications.