Understanding the effects of substrate stiffness on rejuvenation of aging MSCs via single cell analysis.

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Mesenchymal stem cells (MSCs) from bone marrow are widely used in clinical applications due to their therapeutic properties. However, in vitro expansion of MSCs on tissue culture dish (TC) induce aging (replicative senescence), which reduce their quantities and qualities with undefined mechanism.The first-year report demonstrated that the early passage MSCs cultured on hydrogels exhibited the reverse senescence phenotypes. In this second-year report, to confirm the rejuvenation capacity of the hydrogels, the late passage MSCs that already undergone cell cycle arrest were evaluated. In agreement with the results obtained from the young MSCs, down-regulation of senescence markers was also observed on the gels. Treating the MSCs on TC with CSK tension inhibitors downregulated these markers, emphasizing the importance of the CSK tension on cellular senescence. To gain insight into the effect of mechanical stimuli on redox homeostasis, premature senescence was induced using hydrogen peroxide and the cellular stress response was evaluated. On TC, the treated cells increased ROS, antioxidant genes and senescence markers. The hydrogels could attenuate the effects of hydrogen peroxide induced oxidative stress and suppressed senescence, indicating that the MSCs cultured on the hydrogels possess higher radical scavenging capacity and could better maintain redox homeostasis. Knocking down the master of redox regulator enhanced the senescence, suggesting the interconnection between mechanical signaling, redox homeostasis and cellular senescence.