Controlling the senescence of mesenchymal stem cells (MSCs) is essential for improving the efficacy of MSC-based therapies. Here, a model of MSC senescence was established by replicative subculture in tonsil-derived MSCs (TMSCs) using senescence-associated β-galactosidase, telomere-length related genes, stemness, and mitochondrial metabolism. Using transcriptomic and proteomic analyses, we identified glucose-regulated protein 78 (GRP78) as a unique MSC senescence marker. With increasing cell passage number, GRP78 gradually translocated from the cell surface and cytosol to the (peri)nuclear region of TMSCs. A gelatin-based hydrogel releasing a sustained, low level of reactive oxygen species (ROS-hydrogel) was used to improve TMSC quiescence and self-renewal. TMSCs expressing cell surface-specific GRP78 (csGRP78+), collected by magnetic sorting, showed better stem cell function and higher mitochondrial metabolism than unsorted cells. Implantation of csGRP78+ cells embedded in ROS-hydrogel in rats with calvarial defects resulted in increased bone regeneration. Thus, csGRP78 is a promising biomarker of senescent TMSCs, and the combined use of csGRP78+ cells and ROS-hydrogel improved the regenerative capacity of TMSCs by regulating GRP78 translocation.

控制间充质干细胞(MSCs) 的衰老对于提高基于 MSC 的疗法的功效至关重要。在这里，使用衰老相关的 β-半乳糖苷酶、端粒长度相关基因、干性和线粒体代谢，通过在扁桃体来源的 MSC (TMSC) 中进行复制传代培养，建立了 MSC 衰老模型。使用转录组和蛋白质组分析，我们将葡萄糖调节蛋白 78 (GRP78) 鉴定为独特的 MSC 衰老标志物。随着细胞传代次数的增加，GRP78 逐渐从细胞表面和胞质溶胶转移到 TMSCs 的（周）核区。一种基于明胶的水凝胶释放持续的、低水平的活性氧（ROS-水凝胶）被用来改善 TMSC 的静止和自我更新。通过磁分选收集的表达细胞表面特异性 GRP78 (csGRP78+) 的 TMSCs 显示出比未分选细胞更好的干细胞功能和更高的线粒体代谢。在颅骨缺损的大鼠中植入嵌入 ROS 水凝胶中的 csGRP78+ 细胞导致骨再生增加。因此，csGRP78 是一种很有前途的衰老 TMSCs 的生物标志物，并且 csGRP78+ 细胞和 ROS-水凝胶的联合使用改善了通过调节 GRP78 易位来提高 TMSCs 的再生能力。