Rapamycin and nicotinamide treatment attenuates senescence-associated features in mesenchymal stromal cells isolated from elderly donors by modulating autophagy

Mesenchymal stromal cells (MSCs) are promising candidates for regenerative medicine due to their multi-lineage differentiation, immunomodulatory properties, and paracrine effects. Old individuals are the prime target population for cell-based therapies. Donor age significantly hinders the efficacy of autologous cell therapy due to the low quantity and senescent profile of isolated stem cells from aged individuals. Dysregulation of the AMPK-mTOR-autophagy pathway challenges the attenuation of senescence-associated features in aged stem cells. Here, we evaluated the effects of short-term treatment with rapamycin and nicotinamide (NAM) on the attenuation of senescence-associated features in aged MSCs. Aged MSCs were isolated from elderly donors and cultured in the medium supplemented with rapamycin (10 nM) and NAM (5 mM) for the duration of a culture passage. Cell proliferation, expression of CKIs, ROS, senescence-associated changes, senescence-associated secretory phenotype (SASP) profile, and osteogenic differentiation were investigated. Furthermore, AMPK, mTORC1, and mTORC2 activity and level of autophagy were evaluated. Aged MSCs treated with rapamycin and NAM exhibited increased replicative capacity and decreased p16 and p21 expression. In contrast to the senescent profile of aged MSCs, rapamycin, and NAM attenuated the senescence-associated changes, including decreased β-galactosidase expression, dysfunctional lysosomes, reduced total cellular ROS, and improved osteogenic differentiation. Treatment with these compounds reduced pro-inflammatory cytokines, IL-1β and IL-6. These partial reversal of senescence-associated features by rapamycin and NAM were associated with altered AMPK, mTORC1 and mTORC2 activity, and autophagy modulation. Short-term in vitro treatment with rapamycin and NAM can potentially yield high-quality autologous MSCs with improved functional characteristics, possibly improving clinical outcomes of cell-based therapies in the elderly population. These short-term in vitro findings require confirmation in further preclinical studies to assess long-term stability and in vivo relevance.