mTOR inhibition rejuvenates aged canine mesenchymal stem cells by restoring mitochondrial function and telomere integrity, and enhances therapeutic efficacy in atopic dermatitis

Aging-associated dysfunction of mesenchymal stem cells (MSCs) limits the efficacy of regenerative therapies, particularly in autologous treatments for aged patients. Rapamycin, an mTOR inhibitor, has emerged as a promising strategy for delaying stem cell aging, yet its mechanistic effects on epigenetic and metabolic aging pathways in canine MSCs remain unclear. This study, therefore, investigated whether rapamycin-mediated rejuvenation could restore the functional capacity of aged canine adipose-derived MSCs (AMSCs) and improve their therapeutic efficacy in an atopic dermatitis (AD) mouse model. Age-dependent alterations in canine AMSCs, obtained from young (1-year-old, YMSCs) and aged (10-year-old, OMSCs) beagle donors, were characterized, and the rejuvenative potential of short-term, low-dose rapamycin treatment (100 nM, 2 h) was evaluated. Epigenetic modifications were analyzed by profiling DNA methylation patterns using the Horvath mammalian methylation array. Cellular senescence, cell-cycle dynamics, mitochondrial function, telomere integrity, migratory capacity, and immunomodulatory properties were assessed using molecular biology approaches, including qPCR, flow cytometry, Seahorse metabolic analysis, and senescence-associated β-galactosidase (SA-β-gal) staining. The therapeutic effects of rapamycin-treated canine AMSCs were further evaluated in a 2,4-Dinitrochlorobenzene (DNCB)-induced atopic dermatitis model using female BALB/c mice. OMSCs exhibited typical senescence-associated phenotypes, including reduced proliferation, G0/G1 cell-cycle arrest, mitochondrial dysfunction, telomere shortening, and age-associated epigenetic alterations. Rapamycin treatment restored mitochondrial and telomere function, enhanced migratory and immunomodulatory capacities, and attenuated cellular senescence. In the DNCB-induced AD mouse model, rapamycin-rejuvenated OMSCs (ORMSCs) alleviated disease severity, reduced inflammatory responses, serum IgE levels, and Th1/Th2/Th17 and Treg-associated cytokine markers, and restored epidermal barrier gene expression compared with OMSCs. This study demonstrates that rapamycin treatment can reverse multiple hallmarks of aging in aged canine AMSCs, including epigenetic alterations, mitochondrial function, telomere integrity, migratory capacity, and immunomodulatory ability. Furthermore, rapamycin-rejuvenated canine AMSCs showed improved therapeutic efficacy in an AD mouse model by reducing inflammation, restoring immune balance, and repairing epidermal barrier function. Collectively, this study suggests that mTOR inhibition has the potential to improve the quality and therapeutic potential of aged canine AMSCs, providing an avenue for exploration in regenerative and immune therapies in veterinary medicine.