Human endometrium-derived mesenchymal stem cells (hMESCs) enter the premature senescence under sublethal oxidative stress, however underlying mechanism remains unknown. Here, we showed that exogenous H2O2 induces a rapid phosphorylation and co-localization of ATM, H2A.X, 53BP1 leading to DNA damage response (DDR) activation. DDR was accompanied with nuclear translocation of p-p53 followed by up-regulation of p21Waf1 and the permanent hypophosphorylation of pRb. Additionally, the increased p38MAPK/MAPKAPK-2 activation persisted in H2O2-treated cells. We suggest that both p53/p21/pRb and p38MAPK/MAPKAPK-2 pathways are responsible for establishing an irreversible cell cycle arrest that is typical of senescence. The process of further stabilization of senescence required prolonged DDR signaling activation that was provided by the permanent ROS production which in turn was regulated by both p38MAPK and the increased functional mitochondria. To reverse senescence, the pharmacological inhibition of p38MAPK was performed. Cell treatment with SB203580 was sufficient to recover partially senescence phenotype, to block the ROS elevation, to decrease the mitochondrial function, and finally to rescue proliferation. Thus, suppression of the p38MAPK pathway resulted in a partial prevention of H2O2-induced senescence of hMESCs. The current study is the first to reveal the molecular mechanism of the premature senescence of hMESCs in response to oxidative stress.

Cellular senescence was first described as a failure of normal human cells to divide indefinitely in culture. Until recently, the emphasis in the study of cell senescence has been focused on the accompanying intracellular processes. The focus of the attention has been on the irreversible growth arrest and two important physiological functions that rely on it: suppression of carcinogenesis due to the proliferation loss of damaged cells, and the acceleration of organism aging due to the deterioration of the tissue repair mechanism with age. However, the advances of the past years have revealed that senescent cells can impact the surrounding tissue microenvironment, and, thus, that the main consequences of senescence are not solely mediated by intracellular alterations. Recent studies have provided evidence that a pool of molecules secreted by senescent cells, including cytokines, chemokines, proteases and growth factors, termed the senescence-associated secretory phenotype (SASP), via autocrine/paracrine pathways can affect neighboring cells. Today it is clear that SASP functionally links cell senescence to various biological processes, such as tissue regeneration and remodeling, embryonic development, inflammation, and tumorigenesis. The present article aims to describe the social life of senescent cells: basically, SASP constitution, molecular mechanisms of its regulation, and its functional role.

What are the consequences of endometrial stromal cell (EnSC) senescence for endometrial function? Senescence of EnSC contributes to impaired endometrial decidualization and impaired interaction with trophoblast cells but application of senomorphics diminishes the adverse effects of senescent EnSC on decidualization and implantation. A prolonged and highly disordered pro-inflammatory secretory profile of EnSC, which resembles the senescence-associated secretory phenotype, is associated with implantation failure. Furthermore, it has been suggested that implantation failure may be associated with increased EnSC senescence during the proliferative phase of the menstrual cycle. Primary EnSC cell cultures were isolated from endometrial biopsies taken from four patients without any endometrial complications planning to undergo IVF. EnSC senescence was induced by oxidative stress (1 h exposure to 200 µM H₂O₂) followed by 14 days culture but some results were confirmed in a replicative senescence model (after 25 passages). The decidual reaction was evaluated with routine methods and a genetic tool previously designed by us that estimates integral decidual response by fluorescence of a reporter protein. Time-course RNA-sequencing of control and senescent EnSC before and during decidualization was performed using four replicates for each state. To extend our findings, we applied several publicly available datasets. To model implantation in vitro, the choriocarcinoma cell line BeWo b30 was used. To reduce the senescent phenotype of EnSC, two classical senomorphics were applied—rapamycin and metformin. EnSC cultures were used to investigate the effects of senescence on decidualization and on an in vitro implantation model using spheroids derived from BeWo cells. Co-culture models (2D and 3D) were used to explore the effect of senescent cells on neighbouring control cells. The following methods were used to assess cell function, RNA-sequencing, bioinformatic analysis, CRISPR/Cas9 genome editing, FACS, western blotting, RT–PCR, immunofluorescence, molecular cloning, lentiviral transduction and ELISA. Premature senescence of EnSC could be a cause of impaired decidualization. Hormone-induced decidual transformation of EnSC cultures was negatively affected by senescence. Bioinformatics revealed crucial disturbances in the decidual reaction of senescent EnSC which could affect embryo invasion, alter the ‘meta-signature’ of human endometrial receptivity, disturb the emergence of mature and senescent decidual cells subpopulations, impair ligand–receptor interaction with trophoblasts and modify the architecture of extracellular matrix. These predictions were functionally validated using an in vitro implantation model. Moreover, we observed that senescent EnSC, likely via the altered secretome, caused ‘bystander’ quenching of the decidual reaction in adjacent cells, reinforcing dysfunction of the stromal compartment. Application of senomorphics that reduced the senescence phenotype diminished adverse effects of senescent EnSC on decidualization and implantation. The data used in this study are available in the GEO database (GEO identifier GSE160702). The present study was based on in vitro cell cultures derived from only four women. Further studies involving patients with impaired implantation are needed to confirm our findings. The presence of senescent EnSC within the stromal compartment of the endometrium may be a risk-factor for the failure of embryo implantation. Application of senomorphics during the proliferative phase of the menstrual cycle is a promising strategy to alleviate negative effects of senescent EnSC and to improve embryo implantation rates. This study was funded by the Russian Science Foundation (# 19-74-10038). The authors do not have any competing interests to declare.

Intracellular calcium ([Ca2+]i) has been reported to play an important role in autophagy, apoptosis and necrosis, however, a little is known about its impact in senescence. Here we investigated [Ca2+]i contribution to oxidative stress-induced senescence of human endometrium-derived stem cells (hMESCs). In hMESCs sublethal H2O2-treatment resulted in a rapid calcium release from intracellular stores mediated by the activation of PLC/IP3/IP3R pathway. Notably, further senescence development was accompanied by persistently elevated [Ca2+]i levels. In H2O2-treated hMESCs, [Ca2+]i chelation by BAPTA-AM (BAPTA) was sufficient to prevent the expansion of the senescence phenotype, to decrease endogenous reactive oxygen species levels, to avoid G0/G1 cell cycle arrest, and finally to retain proliferation. Particularly, loading with BAPTA attenuated phosphorylation of the main DNA damage response members, including ATM, 53BP1 and H2A.X and reduced activation of the p53/p21/Rb pathway in H2O2-stimulated cells. Next, we revealed that BAPTA induced an early onset of AMPK-dependent autophagy in H2O2-treated cells as confirmed by both the phosphorylation status of AMPK/mTORC1 pathway and the dynamics of the LC3 lipidization. Summarizing the obtained data we can assume that calcium chelation is able to trigger short-term autophagy and to prevent the premature senescence of hMESCs under oxidative stress.

Targeted elimination of senescent cells, senolysis, is one of the core trends in the anti-aging therapy. Cardiac glycosides were recently proved to be a broad-spectrum senolytics. Here we tested senolytic properties of cardiac glycosides towards human mesenchymal stem cells (hMSCs). Cardiac glycosides had no senolytic ability towards senescent hMSCs of various origins. Using biological and bioinformatic approaches we compared senescence development in ‘cardiac glycosides-sensitive’ A549 and ‘-insensitive’ hMSCs. The absence of senolysis was found to be mediated by the effective potassium import and increased apoptosis resistance in senescent hMSCs. Weakening “antiapoptotic defense” predisposes hMSCs to senolysis. We revealed that apoptosis resistance, previously recognized as a common characteristic of senescence, in fact, is not a general feature of senescent cells. Moreover, only apoptosis-prone senescent cells are sensitive to cardiac glycosides-induced senolysis. Thus, we can speculate that the effectiveness of senolysis might depend on whether senescent cells indeed become apoptosis-resistant as compared to their proliferating counterparts. Graphic abstract