Ivanova Julia, Shorokhova Mariia, Pugovkina Natalia, Kozhukharova Irina, Alekseenko Larisa, Guriev Nikita, Kuneev Ivan, Domnina Alisa, Grinchuk Tatiana, Zemelko Victoria, Lyublinskaya Olga
Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretskii pr. 4, St. Petersburg 194064, Russia.
Int J Mol Sci. 2024 Nov 22;25(23):12533. doi: 10.3390/ijms252312533.
Mesenchymal stem/stromal cells (MSCs) are becoming increasingly important for biomedical applications, such as cell therapy, disease modeling, and drug screening. At the same time, long-term cultivation, which is necessary to prepare a sufficient amount of cellular material for therapeutic and research purposes, is accompanied by the development of replicative senescence. Partial reprogramming emerged as a novel method that shows promising results in the rejuvenation of cells in vitro and in vivo; however, it has not yet been applied for human MSCs that have undergone replicative senescence in culture. In the present study, we subjected senescent human endometrial MSCs to partial reprogramming using Sendai virus vectors containing genes encoding Yamanaka transcription factors Oct4, Sox2, Klf4, and c-Myc. Characterization of the MSCs 5 days after transduction showed the loss of key markers of senescence: the youthful morphology was restored, the expression of senescent-associated β-galactosidase and the number of double-strand DNA breaks decreased, proliferation was activated, and the DNA damage response was enhanced. Further, using an in vitro wound-healing assay, we demonstrated that conditioned medium from partially reprogrammed MSCs showed higher therapeutic activity than that from senescent cells. However, a biosafety test revealed the presence of viral components in conditioned medium, which caused the agglutination of erythrocytes. Collectively, our data suggest that partial reprogramming is a potentially effective strategy for the rejuvenation of cultured MSCs in late passages but requires the use of virus-free protocols, such as chemical reprogramming.
间充质干/基质细胞（MSCs）在生物医学应用中变得越来越重要，如细胞治疗、疾病建模和药物筛选。与此同时，为了治疗和研究目的而制备足够数量的细胞材料所必需的长期培养，伴随着复制性衰老的发展。部分重编程作为一种新方法，在体外和体内细胞年轻化方面显示出有前景的结果；然而，它尚未应用于在培养中经历复制性衰老的人MSCs。在本研究中，我们使用含有编码山中转录因子Oct4、Sox2、Klf4和c-Myc基因的仙台病毒载体对衰老的人子宫内膜MSCs进行部分重编程。转导后5天对MSCs的表征显示衰老关键标志物的丧失：恢复了年轻的形态，衰老相关β-半乳糖苷酶的表达和双链DNA断裂的数量减少，增殖被激活，并且DNA损伤反应增强。此外，使用体外伤口愈合试验我们证明，部分重编程的MSCs的条件培养基比衰老细胞的条件培养基显示出更高的治疗活性。然而，生物安全性测试揭示了条件培养基中存在病毒成分，这导致红细胞凝集。总体而言，我们的数据表明部分重编程是晚期传代培养的MSCs年轻化的一种潜在有效策略，但需要使用无病毒方案，如化学重编程。
