ИСТИНА

Adult stem cells are responsible for the replacement of the damaged cells. Unfortunately, these cells do senesce both replicatively and prematurely, what results in their proliferation loss. Indeed, earlier we have shown that human endometrium-derived stem cells (hMESCs) enter the premature senescence in response to sublethal H2O2-treatment. However, senescence is not just a growth arrest: senescent cells secrete a pool of molecules, termed senescence associated secretory phenotype (SASP) that can impact the surrounding tissue microenvironment, what in context of stem cells may have a plenty of side effects. Here, we present evidence that SASP can induce paracrine senescence in normal hMESCs. We revealed that conditioned media (CM) from H2O2-treated hMESCs triggers senescence in their younger counterparts, as indicated by the activation of DNA damage response followed by the initiation of p53/p21 and p16/Rb pathways, leading to reduced proliferation rate. Also we detected other effects of extrinsic factors secreted by senescent cells on naïve hMESCs, such as cell size increase, decreased migration capability, enhanced SA-β-Gal activity, elevated ROS and autofluorescence levels. To further verify these results we established co-cultures of senescent and normal hMESCs. In order to distinguish cells, senescent hMESCs were stained with a fluorescent CFSE dye. In 7 days normal co-cultured hMESCs were sorted for further analysis. Interestingly, normal hMESCs displayed reduced proliferation rate when co-cultured with senescent cells, which confirms a paracrine transmission of senescence. By comparative secretome analysis we identified 92 proteins unique for senescent hMESCs. GO analysis for biological processes of the up-regulated proteins revealed that several enriched processes are involved in the classical SASP response. To validate the results of proteomic analysis we applied Western blotting and ELISA of concentrated CM. Together, we can conclude that hMESCs premature senescence is accompanied by secretion of SASP that is responsible for senescence propagation on the neighboring cells.