Stress-induced premature senescence program is known to be activated in cells by various genotoxic stressors, and oxidative stress is considered to be the main of those. To this end, many studies discover antioxidants as protective anti-aging agents. In the current study, we examined the effects of different antioxidants (Tempol, resveratrol, NAC, DPI) on the mesenchymal stem cells maintained in normal physiological conditions. We used high, but non-cytotoxic antioxidant doses which are widely used in laboratory practice to protect cells from oxidative damage. We show that these substances induce reversible block of cell proliferation and do not cause any genotoxic effects when applied to the quiescent cells. However, the same doses of the same substances, when applied to the proliferating cells, can induce irreversible cell cycle arrest, DNA strand breaks accumulation and DNA damage response activation. As a consequence, antioxidant-induced DNA damage results in the stress-induced premature senescence program activation. We conclude that high doses of antioxidants, when applied to the proliferating cells that maintain physiological levels of reactive oxygen species, can cause DNA damage and induce premature senescence which suggests to re-estimate believed unconditional anti-aging antioxidant properties.

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly, little is known about how quiescent cells respond to environmental challenges. The aim of the present study is to compare stress responses of cycling and quiescent mesenchymal stem cells (MSC). Human endometrial mesenchymal cells (eMSС) were employed as adult stem cells. eMSC quiescence was modeled by serum starvation. Sublethal heat shock (HS) was used as a stress factor. Both quiescent and cycling cells were heated at 45°C for 30 min and then returned to standard culture conditions for their recovery. HS response was monitored by DNA damage response, stress-induced premature senescence (SIPS), cell proliferation activity, and oxidative metabolism. It has been found that quiescent cells repair DNA more rapidly, resume proliferation, and undergo SIPS less than proliferating cells. HS-enforced ROS production in heated cycling cells was accompanied with increased expression of genes regulating redox-active proteins. Quiescent cells exposed to HS did not intensify the ROS production, and genes involved in antioxidant defense were mostly silent. Altogether, the results have shown that quiescent cells are more resistant to heat stress than cycling cells. Next-generation sequencing (NGS) demonstrates that HS-survived cells retain differentiation capacity and do not exhibit signs of spontaneous transformation.

High temperature is a critical environmental and personal factor. Although heat shock is a well-studied biological phenomenon, hyperthermia response of stem cells is poorly understood. Previously, we demonstrated that sublethal heat shock induced premature senescence in human endometrial mesenchymal stem cells (eMSC). This study aimed to investigate the fate of eMSC-survived sublethal heat shock (SHS) with special emphasis on their genetic stability and possible malignant transformation using methods of classic and molecular karyotyping, next-generation sequencing, and transcriptome functional analysis. G-banding revealed random chromosome breakages and aneuploidy in the SHS-treated eMSC. Molecular karyotyping found no genomic imbalance in these cells. Gene module and protein interaction network analysis of mRNA sequencing data showed that compared to untreated cells, SHS-survived progeny revealed some difference in gene expression. However, no hallmarks of cancer were found. Our data identified downregulation of oncogenic signaling, upregulation of tumor-suppressing and prosenescence signaling, induction of mismatch, and excision DNA repair. The common feature of heated eMSC is the silence of MYC, AKT1/PKB oncogenes, and hTERT telomerase. Overall, our data indicate that despite genetic instability, SHS-survived eMSC do not undergo transformation. After long-term cultivation, these cells like their unheated counterparts enter replicative senescence and die.

Temperature is an important exogenous factor capable of leading to irreversible processes in the vital activity of cells. However, the long-term effects of heat shock (HS) on mesenchymal stromal cells (MSC) remain unstudied. We investigated the karyotype and DNA repair drivers and pathways in the human endometrium MSC (eMSC) survived progeny at passage 6 after sublethal heat stress (sublethal heat stress survived progeny (SHS-SP)). G-banding revealed an outbreak of random karyotype instability caused by chromosome breakages and aneuploidy. Molecular karyotyping confirmed the random nature of this instability. Transcriptome analysis found homologous recombination (HR) deficiency that most likely originated from the low thermostability of the AT-rich HR driving genes. SHS-SP protection from transformation is provided presumably by low oncogene expression maintained by tight co-regulation between thermosensitive HR drivers BRCA, ATM, ATR, and RAD51 (decreasing expression after SHS), and oncogenes mTOR, MDM2, KRAS, and EGFR. The cancer-related transcriptomic features previously identified in hTERT transformed MSC in culture were not found in SHS-SP, suggesting no traits of malignancy in them. The entrance of SHS-SP into replicative senescence after 25 passages confirms their mortality and absence of transformation features. Overall, our data indicate that SHS may trigger non-tumorigenic karyotypic instability due to HR deficiency and decrease of oncogene expression in progeny of SHS-survived MSC. These data can be helpful for the development of new therapeutic approaches in personalized medicine.

The study of ion channels in stem cells provides important information about their role in stem cell fate. Previously we have identified the activity of calcium-activated potassium channels of big conductance (BK channels) in human endometrium-derived mesenchymal stem cells (eMSCs). BK channels could have significant impact into signaling processes by modulating membrane potential. The membrane potential and ionic permeability dynamically changes during cycle transitions. Here, we aimed at verification of the role of BK channels as potassium transporting pathway regulating cell cycle passageway of eMSCs. The functional expression of native BK channels was confirmed by patch-clamp and immunocytochemistry. In non-synchronized cells immunofluorescent analysis revealed BK-positive and BK-negative stained eMSCs. Using cell synchronization, we found that the presence of BK channels in plasma membrane was cell cycle-dependent and significantly decreased in G2M phase. However, the study of cell cycle progression in presence of selective BK channel inhibitors showed no effect of pore blockers on cycle transitions. Thus, BK channel-mediated K+ transport is not critical for the fundamental mechanism of passageway through cell cycle of eMSCs. At the same time, the dynamics of the presence of BK channels on plasma membrane of eMSCs can be a novel indicator of cellular proliferation.