Quantitative assessment of changes in cellular morphology at photodynamic treatment in vitro by means of digital holographic microscopy.

Temporal dependence of changes in the morphological characteristics of cells of two cultured lines of cancer origin, HeLa and A549, induced by photodynamic treatment with Radachlorin photosensitizer, have been monitored using digital holographic microscopy during first two hours after short-term irradiation. The observed post-treatment early dynamics of the phase shift in the transmitted wavefront indicated several distinct scenarios of cell behavior depending upon the irradiation dose. In particular the phase shift increased at low doses, which can be associated with apoptosis, while at high doses it decreased, which can be associated with necrosis. As shown, the two cell types responded differently to similar irradiation doses. Although the sequence of death scenarios with the increase of the irradiation dose was the same, each scenario was realized at substantially different doses. These findings suggest that the average phase shift of the transmitted wavefront can be used for quantitative non-invasive cell death characterization. The conclusions made were cofirmed by commonly used test assays using confocal fluorescent microscopy.

High doses of synthetic antioxidants induce premature senescence in cultivated mesenchymal stem cells.

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.

Flow cytometric HyPer-based assay for hydrogen peroxide.

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H2O2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H2O2, by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H2O2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H2O2-reactive dye H2DCFDA and, contrary to the H2DCFDA-based assay, can be employed for the kinetic studies of H2O2 utilization by cells, including measurements of the rate constants of H2O2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H2O2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H2O2.

"Social Life" of Senescent Cells: What Is SASP and Why Study It?

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.

Quiescent Human Mesenchymal Stem Cells Are More Resistant to Heat Stress than Cycling Cells.

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.

Redox environment in stem and differentiated cells: A quantitative approach.

Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa) maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

Molecular Genetic Analysis of Human Endometrial Mesenchymal Stem Cells That Survived Sublethal Heat Shock.

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.

ROLE OF P38 MAP-KINASE IN THE STRESS-INDUCED SENESCENCE PROGRESSION OF HUMAN ENDOMETRIUM-DERIVED MESENCHYMAL STEM CELLS.

Our recent findings clearly demonstrate that human endometrium-derived mesenchymal stem cells (hMESCs) respond to the sublethal oxidative stress by the premature senescence induction via ÀÒÌ/Chk2/p53/ p21/Rb pathway. Furthermore, based on the application of the SB203580 (SB) we suggested p38 MAP-kinase involvement in senescence progression. However, there are several disadvantages concerning this inhibitor: 1) using SB would not be suitable for in vivo experiments due to toxicity issue; 2) the poor kinase selectivity profile of SB complicates interpretation of the obtained data. Here, in order to confirm the implication of p38 in the H2O2-induced senescence of hMESCs, we applied another highly specific inhibitor of p38 ­ BIRB796 (BIRB). In presence of BIRB we revealed cell size decrease, reduction in the levels of reactive oxygen species, partial restoration of proliferation and increase in Rb phosphorylation levels in comparison to H2O2-treated hMESCs. Summarizing the obtained results we can postulate p38 implication in H2O2-induced senescence of hMESCs, and suggest p38 inhibition as a promising approach in prevention of premature senescence.

[PRODUCTION AND CHARACTERIZATION OF THE PANEL OF MONOCLONAL ANTIBODIES AGAINST HUMAN ENDOGLIN].

Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-ß, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.

Reactive Oxygen Species Are Required for Human Mesenchymal Stem Cells to Initiate Proliferation after the Quiescence Exit.

The present study focuses on the involvement of reactive oxygen species (ROS) in the process of mesenchymal stem cells "waking up" and entering the cell cycle after the quiescence. Using human endometrial mesenchymal stem cells (eMSCs), we showed that intracellular basal ROS level is positively correlated with the proliferative status of the cell cultures. Our experiments with the eMSCs synchronized in the G0 phase of the cell cycle revealed a transient increase in the ROS level upon the quiescence exit after stimulation of the cell proliferation. This increase was registered before the eMSC entry to the S-phase of the cell cycle, and elimination of this increase by antioxidants (N-acetyl-L-cysteine, Tempol, and Resveratrol) blocked G1-S-phase transition. Similarly, a cell cycle arrest which resulted from the antioxidant treatment was observed in the experiments with synchronized human mesenchymal stem cells derived from the adipose tissue. Thus, we showed that physiologically relevant level of ROS is required for the initiation of human mesenchymal stem cell proliferation and that low levels of ROS due to the antioxidant treatment can block the stem cell self-renewal.

[INDUCTION OF DECIDUAL DIFFERENTIATION OF ENDOMETRIAL MESENCHYMAL STEM CELLS].

In this study, we compared the ability of human mesenchymal stem cells derived from menstrual blood (eMSCs) and mesenchymal stem cells (MSCs) from other tissues to differentiate into decidual cells in vitro. It was demonstrated that during differentiation secretion of decidualization markers (prolactin and insulin-like growth factor binding protein-1) increases in eMSCs from adipose tissue (MSC-AD). Thus, the ability of eMSCs to differentiate into decidual cells is much higher than MSC-BM or MSC-AD. It makes eMSCs promising for application in cellular therapy of infertility associated with decidualzation insufficiency.

[PAIRWISE CROSS-SPECIES TRANSCRIPTOME ANALYSIS OF POLYPLOIDY-ASSOCIATED EXPRESSION CHANGES OF DEVELOPMENTAL GENE MODULES].

Design and development of highly sensitive method bioinformatics are important for investigation of casual relationships between epigenetic changes and gene activity. Cell polyploidy may trigger such changes. However, maintaining the balance of gene dosage, polyploidy may provide only a rather weak effect on their expression. Currently, there is no comprehensive and concordant data in regard to ploidy-associated transcriptomic changes. To find out how polypoidy affects gene activity, we have developed an integrative bioinformatic method of pairwise cross-species transcriptome analysis of mammalian tissues with various polyploidy degrees. The main benefit of this approach is its ability to separate species- and tissue-specific noises of evolutionary conserved effects. We demonstrat the application of the method for the analysis of gene modules and protein interactions networks coordinating programs of development, differentiation and pluripotency. The analysis was performed with transcriptomes of polyploid and diploid organs (human and mouse heart and liver). Our data indicate that ploidy-induced genes enrich Gene Ontology (GO) biological processes and KEGG pathways related to development, morphogenesis and stem cells biology (including Hippo, Pi3K, WNT, Hedgehog and TGF-ß pathways) with higher degree than ploidy-inhibited genes. Thas, our data are the first to show that polyploidy may induce and coordinate developmental modules.

[CHARACTERISTIC OF ENDOMETRIAL MESENCHYMAL STEM CELLS IN CULTURE OBTAINED FROM PATIENT WITH ADENOMYOSIS].

Adenomyosis is form of endometriosis, common diseases of female reproductive system, which can lead to infertility in women. in this study we are obtained and characterized cell line endometrial mesenchymal stem cells from a patient with adenomyosis, and compare obtained cells with the cell line of healthy donor. Aim of this study was to assesses the extent of differences between cells from donor with adenomyosis and cells from healthy donor. Was established that compared lines had morphology like fibroblasts, were differentiated in adipocytes, were expressed mesenchymal markers and didn't expressed haematopoietic markers. Cytogenetic analysis of differentially stained metaphase chromosomes on G-banding (passage 6-7) showed that healthy donor's cells had predominantly normal karyotype. The cellular line from a patient with diagnosis of "adenomyosis" had a lot of cells with changes in karyotype's structure. These changes were related with aneuploidy of cellular population and the presence non-random chromosomal breaks, often in chromosomes 7 and 11. Analysis of this data allows the cells from adenomyosis characterized physiological stability in culture and karyotypic instability with non-random involvement certain chromosomal set. The cellular line obtained from donor with adenomyosis showed signs destabilization of he genome, typical for cell transformation. Division of adenomyosis cells to the 26th passage is stopped and these cells entered into a phase of replicative aging. Based on this, we can conclude that founded karyotype's hanges do not lead to transformation and immortalization of cells in vitro.

[RELATIONSHIP BETWEEN p53/p21/Rb AND MAPK SIGNALING PATHWAYS IN HUMAN ENDOMETRIUM-DERIVED STEM CELLS UNDER OXIDATIVE STRESS].

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H2O2 activate both p53/p21/Rb and p38MAPK/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However the mutual relations between p53/p21/Rb and MAPK signaling pathways, including ERK, p38 and JNK remain unexplored as yet. Here, we used the specific inhibitors--pifithrin-α (PFT), U0126, SB203580 and SP600125 to "switch off" one of the proteins in these cascades and to evaluate the functional status alterations of the rest proteins. Suppression each of the MAPK significantly increased the p53 phosphorylation levels, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation compared with p38 and JNK. These results suppose the existence of the reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. Analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as the ERK antagonists: JNK is capable to activate ERK, while p38 may block the ERK activation. Together, these results demonstrate complex links between different signaling cascades in stressed hMESC, implicating ERK, p38 and JNK in regulation of the premature senescence via p53/p21/Rb pathway.

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products.

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

Disruption of the actin cytoskeleton leads to inhibition of mitogen-induced cyclin E expression, Cdk2 phosphorylation, and nuclear accumulation of the retinoblastoma protein-related p107 protein.

The actin cytoskeleton has been found to be required for mitogen-stimulated cells to passage through the cell cycle checkpoint. Here we show that selective disruption of the actin cytoskeleton by dihydrocytochalasin B (H(2)CB) blocked the mitogenic effect in normal Swiss 3T3 cells, leading to cell cycle arrest at mid to late G(1) phase. Cells treated with H(2)CB remain tightly attached to the substratum and respond to mitogen-induced MAP kinase activation. Upon cytoskeleton disruption, however, growth factors fail to induce hyperphosphorylation of the retinoblastoma protein (pRb) and the pRb-related p107. While cyclin D1 induction and cdk4-associated kinase activity are not affected, induction of cyclin E expression and activation of cyclin E-cdk2 complexes are greatly inhibited in growth-stimulated cells treated with H(2)CB. The inhibition of cyclin E expression appears to be mediated at least in part at the RNA level and the inhibition of cdk2 kinase activity is also attributed to the decrease in cdk2 phosphorylation and proper subcellular localization. The expression patterns of cdk inhibitors p21 and p27 are similar in both untreated and H(2)CB-treated cells upon serum stimulation. In addition, the changes in subcellular localization of pRb and p107 appear to be linked to their phosphorylation states and disruption of normal actin structure affects nuclear migration of p107 during G(1)-to-S progression. Taken together, our results suggest that the actin cytoskeleton-dependent G(1) arrest is linked to the cyclin-cdk pathway. We hypothesize that normal actin structure may be important for proper localization of certain G(1) regulators, consequently modulating specific cyclin and kinase expression.

Intracellular distribution of phospholipase C gamma 1 in cell lines with different levels of transformation.

The intracellular localization of phospholipase C gamma 1 (PLC gamma 1) was studied in cell lines with different levels of cell transformation. Immunofluorescence analysis of cell lines with differently organized actin cytoskeletons (A431 cells, HeLa cells, mouse hepatoma MH 22A, Zajdela ascitic hepatoma, primary human embryo skin and lung fibroblasts) gave evidence that PLC gamma 1 is colocalized only with cortical actin and not with stress fibers. Coimmunoprecipitation experiments indicated that PLC gamma 1 was bound to actin in all cell lines investigated. Further, the nuclei of highly transformed cell lines (A431 cells, HeLa cells, mouse hepatoma MH 22A, rat Zajdela ascitic hepatoma) were labeled with the anti-PLC gamma 1 antibody. In contrast, PLC gamma 1 was not observed in the nuclei of primary human embryo skin or lung fibroblasts. Since PLC gamma 1 exists only in the nuclei of highly transformed cell lines, we propose that the distinctive intracellular localization of PLC gamma 1 in normal and highly transformed cell lines may reflect differences in cell signaling systems and mitogenic cell signal transduction.