SLFN11 binds DDX1 to counteract human mesenchymal stem/stromal cell senescence through inhibition of mTOR/eIF4E signaling pathway activation.
AIMS Mesenchymal stem/stromal cells (MSCs) are indispensable for cellular therapy; however, long-term in vitro expansion triggers replicative and stress-induced senescence, which undermines their stemness and therapeutic potential. This study aimed to investigate the protective effect and underlying mechanism of the Schlafen (SLFN) 11 gene against human MSC senescence in vitro and to evaluate the efficacy and safety of SLFN11-overexpressing MSCs. MATERIALS AND METHODS SLFN11 was overexpressed in human MSCs via lentiviral transduction. Protective effects were comprehensively assessed in replicative and stress-induced senescence models by evaluating cell cycle arrest, proliferative and clonogenic capacities, DNA damage, pluripotency, and β-galactosidase activity. Molecular interactions were elucidated through co-immunoprecipitation (Co-IP) and LC-MS/MS, whereas therapeutic efficacy was determined using a DSS-induced ulcerative colitis mouse model. KEY FINDINGS SLFN11 overexpression significantly attenuated senescence by reducing DNA damage accumulation, reversing cell cycle arrest, and enhancing proliferation, clonogenicity, and stemness properties. Mechanistically, SLFN11 bound the RNA helicase DDX1 to attenuate mTOR/eIF4E signaling, thereby resisting senescence. Compared to control MSCs, SLFN11-overexpressing MSCs exhibited superior protection against ulcerative colitis, as evidenced by reduced colonic lesions, maintenance of mucosal integrity, and inhibition of proinflammatory factor secretion. SLFN11-overexpressing MSCs also showed no detectable tumorigenicity or chromosomal aberrations. SIGNIFICANCE These findings delineate a non-canonical role for SLFN11 safeguarding MSCs from senescence via the DDX1/mTOR/eIF4E axis. SLFN11-overexpressing MSCs therefore constitute a promising and safe cell therapy source, characterized by preserved pluripotency and augmented therapeutic efficacy.