Background: Mesenchymal stem cells (MSCs) have potential for treating degenerative and immune diseases, but their clinical efficacy is limited by senescence, characterized by mitochondrial dysfunction, impaired mitophagy, and metabolic imbalance. The goal of this study was to investigate the effects of dimethyloxalylglycine (DMOG), a hypoxia-mimetic agent that stabilizes hypoxia-inducible factor 1 alpha (HIF-1α), on rejuvenating senescent MSCs by enhancing mitochondrial function, mitophagy, and metabolic reprogramming.
Methods: Two models of MSC senescence were established: oxidative stress-induced senescence using hydrogen peroxide and replicative senescence through serial passaging. Umbilical cord derived MSCs were treated with DMOG for 48 h under normoxic conditions. Mitochondrial function, mitophagy, and metabolism were assessed using assays that measured mitochondrial membrane potential, reactive oxygen species levels, ATP production, and mitophagy. Western blotting and real-time PCR were employed to analyze the expression changes of relevant molecules. RNA sequencing (RNA-seq) was performed to identify key genes and pathways regulated by DMOG. Additionally, to evaluate the therapeutic potential of rejuvenated MSCs, a co-culture system was established, where DMOG-treated senescent MSCs were co-cultured with IL-1β-treated chondrocytes.
Results: DMOG treatment significantly reduced key senescence markers, including senescence-associated beta-galactosidase, p53, and p21, in both senescence models. DMOG treatment restored mitochondrial morphology and function, improving mitochondrial membrane potential, reducing mitochondrial reactive oxygen species, and enhancing ATP production. DMOG also promoted mitophagy, as evidenced by increased colocalization of mitochondria with lysosomes. RNA-seq analysis revealed that DMOG activated key pathways, including HIF-1 signaling, calcium signaling, and mitophagy-related gene (BNIP3 and BNIP3L). Notably, BNIP3 knockdown greatly abolished DMOG-induced mitophagy and its anti-senescence effects. Furthermore, DMOG treatment improved metabolic flexibility by enhancing both mitochondrial respiration and glycolysis in senescent MSCs. Moreover, DMOG-treated senescent MSCs partially restored their therapeutic efficacy in an osteoarthritis model by improving extracellular matrix regulation in IL-1β-stimulated chondrocytes.
Conclusions: Short-term DMOG treatment rejuvenates senescent MSCs by enhancing mitochondrial function, promoting mitophagy via HIF-1α/BNIP3, and improving metabolic reprogramming. DMOG-treated MSCs also showed enhanced therapeutic efficacy in co-culture with IL-1β-treated chondrocytes, suggesting its potential to improve MSC-based therapies in regenerative medicine.
Keywords: BNIP3; HIF-1α; Hypoxia-Mimetic agent; Mesenchymal stem cells (MSCs); Mitophagy; Senescence.
© 2025. The Author(s).