Olga Lyublinskaya

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.

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated β-galactosidase (SA-β-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72xa0h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity.

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.

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.

Mesenchymal stem cells with irreversibly arrested proliferation stimulate decidua development in rats

Stem cell transplantation, which is based on the application of mesenchymal stem/stromal cells (MSCs), is a rapidly developing approach to the regenerative therapy of various degenerative disorders characterized by brain and heart failure, as well as skin lesions. In comparison, the use of stem cell transplantations to treat infertility has received less attention. One of the causes of miscarriages and fetal growth delay is the loss of the decidual reaction of endometrial cells. The present study modeled decidualization processes in pseudopregnant rats. For cell transplantation experiments, the rats were transplanted with MSCs established from endometrial fragments in menstrual blood (eMSCs). These cells express common MSC markers, are multipotent and are able to differentiate into various tissue lineages. Cell therapy frequently requires substantial cell biomass, and cultivation of MSCs may be accompanied by significant changes to their properties, including malignant transformation. In order to minimize the potential for malignant transformation, the proliferation of eMSCs was irreversibly suppressed by irradiation and mitomycin C treatment. Transplantation of the rats with viable, non-proliferating eMSCs stimulated the development of all elements of decidual tissue. Conversely, transplantation of the rats with cells killed using 95% ethanol did not result in the development of decidual tissue. The present study demonstrated the potential for applying eMSCs to the cellular therapy of infertility associated with endometrial disorders characterized by decidualization insufficiency and implantation failure. In addition, the transplantation of viable but non-proliferating cells ensured that their oncogenic potential was limited.

The effect of yeast Saccharomyces cerevisiae red pigment on the expression of cloned human α-synuclein

Two transgenic yeast strains expressing human α-synuclein were used to study the impact of yeast red pigment exhibiting antiamyloid properties. It has been demonstrated that the endogenous red pigment produced under special conditions in strains carrying an ade1 mutation inhibits the expression of the hybrid protein α-synuclein-GFP. This was evident from the reduced mean value of GFP fluorescence and diminished number of cells accumulating cytoplasmic inclusions of α-synuclein-GFP. Exogenous forms of the purified red pigment (natural, synthetic and hydrolyzed derivatives) differ from the endogenous red pigment by their effect on α-synuclein. Exogenous red pigments increased the number of both cells expressing GFP fluorescence and those containing cytoplasmic inclusions. However, both endogenous and exogenous red pigments reduced the cloned α-synuclein toxicity and resulted in redistribution of the α-synuclein in cells. α-Synuclein content decreased in cell lysate pellets and increased in supernatants.

Human mesenchymal stem cells in spheroids improve fertility in model animals with damaged endometrium

BackgroundAsherman’s syndrome (AS) is one of the gynecological disorders caused by the destruction of the endometrium. For some cases of AS available surgical methods and hormonal therapy are ineffective. Stem cell transplantation may offer a potential alternative for AS cure.MethodsHuman endometrial mesenchymal stem cells (eMSC) organized in spheroids were transplanted in rats with damaged endometrium modeled on AS. Treatment response was defined as pregnancy outcome and litter size.ResultsApplication of eMSC in spheroids significantly improved the rat fertility with the AS model. eMSC organized in spheroids retain all properties of eMSC in monolayer: growth characteristics, expression of CD markers, and differentiation potential. Synthesis of angiogenic and anti-inflammatory factors drastically increased in eMSC assembled into spheroids.ConclusionsHuman endometrial mesenchymal stem cells (eMSC) can be successfully applied for Asherman’s syndrome (AS) treatment in the rat model. eMSC organized in spheroids were more therapeutically effective than the cells in monolayer. After transplantation of eMSC in spheroids the pregnancy outcome and litter size in rats with AS was higher than in rats that received autologous rat bone marrow cells. It suggests the therapeutic plausibility of heterologous eMSC in case of failure to use autologous cells.

Therapeutic doses of doxorubicin induce premature senescence of human mesenchymal stem cells derived from menstrual blood, bone marrow and adipose tissue

Doxorubicin (Dox) is an effective anticancer drug with known activity against a wide spectrum of malignancies, hematologic malignancies in particular. Despite extensive clinical use, the mechanisms of its side effects and negative action on normal cells remain under study. The aim of this study was to investigate the effect of Dox on cultured human mesenchymal stem cells (MSCs) derived from menstrual blood (eMSCs), bone marrow (BMSCs) and adipose tissue (AMSCs). Dox treatment in high doses decreased the survival of MSCs in a dose-dependent manner. Clinically relevant low doses of Dox induced premature senescence of eMSCs, BMSCs and AMSCs, but did not kill the cells. Dox caused cell cycle arrest and formation of γ-H2AX foci, and increased the number of SA-β-gal-positive cells. BMSCs entered premature senescence earlier than other MSCs. It has been reported that neural-like cells differentiated from MSCs of various origins are more sensitive to Dox than their parent cells. Dox-treated differentiated MSCs exhibited lower viability and earlier generation of γ-H2AX foci. Dox administration inhibited secretory activity in neural-like cells. These findings suggest that a clinically relevant Dox dose damages cultured MSCs, inducing their premature senescence. MSCs are more resistant to this damage than differentiated cells.

Flow cytometric HyPer-based assay for hydrogen peroxide.

ABSTRACT 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. Graphical abstract Figure. No Caption available. HighlightsRatiometric flow cytometry (FC) analysis is applicable for HyPer‐based studies.FC is suitable to detect submicromolar concentrations of extracellularly added H2O2.FC can be employed for the measurements of the rate constants of H2O2 removal.FC is suitable to detect changes in the level of endogenously generated H2O2.Multi‐parameter FC analysis enables detection of H2O2 in different cell populations.

Oxidative stress response of human fibroblasts and endometrial mesenchymal stem cells

Human mesenchymal stem cells are a promising cell source for tissue engineering. During transplantation, they may be subjected to oxidative stress due to unfavorable cellular microenvironment characterized by an increased level of reactive oxygen species. Recently, we have demonstrated that oxidative stress response of human mesenchymal stem cells derived from endometrium (hMESCs) depends on the oxidizer concentration. The duration of cell treatment with an oxidizer also may play an important role. In this study, we investigated the dependence of the cell response on H2O2 treatment duration. The effects of high H2O2 doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H2O2 treatment for 60 min caused multiphase cell cycle arrest, with dose-dependent cell death occurring equally in all phases of the cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H2O2 treatment from 60 to 10 min induced growth retardation, G1-phase cell accumulation, and cell size increase. Collectively, these findings suggest that there is induction of premature senescence. Thus, shortening of oxidative stress induced in human endometrial stem cells and embryonic fibroblasts by high H2O2 doses enables one to modulate cellular response as both cell death and premature senescence.