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

骨髓间充质干细胞（Mesenchymal stem cells，MSCs）因其治疗特性而广泛应用于临床。然而，MSCs在组织培养皿（tissue culture dish，TC）上的体外扩增导致了细胞的衰老（replicative senescence，复制性衰老），导致细胞数量和质量的下降，其机制尚不清楚。在第二年的报告中，为了确认水凝胶的复壮能力，对已经经历细胞周期停滞的晚期传代MSC进行了评价。与从年轻MSC获得的结果一致，在凝胶上也观察到衰老标志物的下调。用CSK张力抑制剂处理TC上的MSC下调了这些标志物，强调了CSK张力对细胞衰老的重要性。为了深入了解机械刺激对氧化还原稳态的影响，使用过氧化氢诱导过早衰老，并评估细胞应激反应。TC处理的细胞增加了ROS、抗氧化基因和衰老标志物。该水凝胶可减弱过氧化氢诱导的氧化应激的影响，并抑制衰老，表明在水凝胶上培养的MSC具有更高的自由基清除能力，并能更好地维持氧化还原稳态。敲除氧化还原调节因子的主导因子可增强衰老，提示机械信号、氧化还原稳态与细胞衰老之间存在着内在联系。

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.