Application of genetically encoded biosensors of redox-active compounds promotes the elaboration of new methods for investigation of intracellular redox activities. Previously, we have developed a method to measure quantitatively the intracellular concentration of hydrogen peroxide (H₂O₂) in living cells using genetically encoded biosensor HyPer. In the present study, we refined the method and applied it for comparing the antioxidant system potency in human cells of different phenotypes by measuring the gradient between the extracellular and cytoplasmic H₂O₂ concentrations under conditions of H₂O₂-induced external oxidative stress. The measurements were performed using cancer cell lines (K-562 and HeLa), as well as normal human cells – all expressing HyPer in the cell cytoplasm. As normal cells, we used three isogenic lines of different phenotypes – mesenchymal stem/stromal cells (MSCs), induced pluripotent stem cells (iPSCs) derived from MSCs by reprogramming, and differentiated iPSC progenies with the phenotype resembling precursory MSCs. When exposing cells to exogenous H₂O₂, we showed that at low oxidative loads (50 μM of H₂O₂), which caused the exhaustion of thioredoxin activity in the cell cytoplasm, the gradient stabilized, pointing out that it is the functional status of the thioredoxin-depended enzymatic system that drives the dependence of the H₂O₂ gradient on the oxidative load in human cells. At high H₂O₂ concentrations, the cytoplasmic H₂O₂ level in cancer cells was found to be several hundred times lower than the extracellular one. At the same time, in normal cells, extracellular-to-intracellular gradient amounted to thousands of times. Upon reprogramming, the potency of cellular antioxidant defense increased. In contrast, differentiation of iPSCs did not result in the changes in antioxidant system activity in the cell cytoplasm, assuming that intensification of the H₂O₂-detoxification processes is inherent to a period of early human development.

Hormone-regulated proliferation and differentiation of endometrial stromal cells (ESCs) determine overall endometrial plasticity and receptivity to embryos. Previously we revealed that ESCs may undergo premature senescence, accompanied by proliferation loss and various intracellular alterations. Here we focused on whether and how senescence may be transmitted within the ESCs population. We revealed that senescent ESCs may induce paracrine senescence in young counterparts via cell contacts, secreted factors and extracellular vesicles. According to secretome-wide profiling we identified plasminogen activator inhibitor -1 (PAI-1) to be the most prominent protein secreted by senescent ESCs (data are available via ProteomeXchange with identifier PXD015742). By applying CRISPR/Cas9 techniques we disclosed that PAI-1 secreted by senescent ESCs may serve as the master-regulator of paracrine senescence progression within the ESCs population. Unraveled molecular basis of senescence transduction in the ESCs population may be further considered in terms of altered endometrial plasticity and sensitivity to invading embryo, thus contributing to the female infertility curing.

Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment. Mechanosensitive channels are part of the cellular toolkit that feels the mechanical environment and can transduce mechanical stimuli to intracellular signaling pathways. Here, we identify previously recorded, mechanosensitive (MS), stretch-activated channels as Piezo1 proteins in the plasma membrane of eMSCs. Piezo1 activity triggered by the channel agonist Yoda1 elicits influx of Ca²⁺, a known modulator of cytoskeleton reorganization and cell motility. We found that store-operated Ca²⁺ entry (SOCE) formed by Ca²⁺-selective channel ORAI1 and Ca²⁺ sensors STIM1/STIM2 contributes to Piezo1-induced Ca²⁺ influx in eMSCs. Particularly, the Yoda1-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) is partially abolished by 2-APB, a well-known inhibitor of SOCE. Flow cytometry analysis and wound healing assay showed that long-term activation of Piezo1 or SOCE does not have a cytotoxic effect on eMSCs but suppresses their migratory capacity and the rate of cell proliferation. We propose that the Piezo1 and SOCE are both important determinants in [Ca²⁺]_i regulation, which critically affects the migratory activity of eMSCs and, therefore, could influence the regenerative potential of these cells.

Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. Significance: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.