The Role of Intracellular Potassium in Cell Quiescence, Proliferation, and Death.

This brief review explores the role of intracellular K+ during the transition of cells from quiescence to proliferation and the induction of apoptosis. We focus on the relationship between intracellular K+ and the growth and proliferation rates of different cells, including transformed cells in culture as well as human quiescent T cells and mesenchymal stem cells, and analyze the concomitant changes in K+ and water content in both proliferating and apoptotic cells. Evidence is discussed indicating that during the initiation of cell proliferation and apoptosis changes in the K+ content in cells occur in parallel with changes in water content and therefore do not lead to significant changes in the intracellular K+ concentration. We conclude that K+, as a dominant intracellular ion, is involved in the regulation of cell volume during the transit from quiescence, and the content of K+ and water in dividing cells is higher than in quiescent or differentiated cells, which can be considered to be a hallmark of cell proliferation and transformation.

Accumulation of Dystrophin-Positive Muscle Fibers and Improvement of Neuromuscular Junctions in mdx Mouse Muscles after Bone Marrow Transplantation under Different Conditions.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder caused by mutations in the dystrophin gene. It leads to respiratory and cardiac failure and premature death at a young age. Although recent studies have greatly deepened the understanding of the primary and secondary pathogenetic mechanisms of DMD, an effective treatment remains elusive. In recent decades, stem cells have emerged as a novel therapeutic product for a variety of diseases. In this study, we investigated nonmyeloablative bone marrow cell (BMC) transplantation as a method of cell therapy for DMD in an mdx mouse model. By using BMC transplantation from GFP-positive mice, we confirmed that BMCs participate in the muscle restoration of mdx mice. We analyzed both syngeneic and allogeneic BMC transplantation under different conditions. Our data indicated that 3 Gy X-ray irradiation with subsequent BMC transplantation improved dystrophin synthesis and the structure of striated muscle fibers (SMFs) in mdx mice as well as decreasing the death rate of SMFs. In addition, we observed the normalization of neuromuscular junctions (NMJs) in mdx mice after nonmyeloablative BMC transplantation. In conclusion, we demonstrated that nonmyeloablative BMC transplantation could be considered a method for DMD treatment.

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis.

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed.

Paracrine and Autocrine Effects of VEGF Are Enhanced in Human eMSC Spheroids.

The mechanisms underlying the therapeutic potential of MSCs are the focus of intense research. We studied human MSCs isolated from desquamated endometrium (eMSCs), which, as previously shown, have high regenerative potential in various disease models. The aim was to evaluate the role of secreted VEGF in stimulating angiogenesis and maintaining eMSC viability and migration, which is important for improving the therapeutic properties of MSCs. We compared three eMSC cultures differing in the level of VEGF secretion: 3D spheroids, monolayer eMSCs, and monolayer eMSCs with VEGF knockdown. Spheroid eMSCs produced higher amounts of VEGF and had the strongest paracrine effect on HUVEC. eMSCs with VEGF knockdown did not stimulate angiogenesis. Monolayered eMSCs expressed VEGFR1, while spheroid eMSCs expressed both VEGFR1 and VEGFR2 receptors. The knockdown of VEGF caused a significant decrease in the viability and migration of eMSCs. eMSCs from 3D spheroids enhanced proliferation and migration in response to exogenous VEGF, in contrast to monolayered eMSCs. Our results suggest that the VEGF-VEGFR1 loop appears to be autocrine-involved in maintaining the viability of eMSCs, and VEGFR2 expression enhances their response to exogenous VEGF, so the angiogenic potential of eMSC can be up- or downregulated by intrinsic VEGF signals.

Monovalent ions and stress-induced senescence in human mesenchymal endometrial stem/stromal cells.

Monovalent ions are involved in growth, proliferation, differentiation of cells as well as in their death. This work concerns the ion homeostasis during senescence induction in human mesenchymal endometrium stem/stromal cells (hMESCs): hMESCs subjected to oxidative stress (sublethal pulse of H2O2) enter the premature senescence accompanied by persistent DNA damage, irreversible cell cycle arrest, increased expression of the cell cycle inhibitors (p53, p21) cell hypertrophy, enhanced ß-galactosidase activity. Using flame photometry to estimate K+, Na+ content and Rb+ (K+) fluxes we found that during the senescence development in stress-induced hMESCs, Na+/K+pump-mediated K+ fluxes are enhanced due to the increased Na+ content in senescent cells, while ouabain-resistant K+ fluxes remain unchanged. Senescence progression is accompanied by a peculiar decrease in the K+ content in cells from 800-900 to 500-600 µmol/g. Since cardiac glycosides are offered as selective agents for eliminating senescent cells, we investigated the effect of ouabain on ion homeostasis and viability of hMESCs and found that in both proliferating and senescent hMESCs, ouabain (1 nM-1 µM) inhibited pump-mediated K+ transport (ID50 5 × 10-8 M), decreased cell K+/Na+ ratio to 0.1-0.2, however did not induce apoptosis. Comparison of the effect of ouabain on hMESCs with the literature data on the selective cytotoxic effect of cardiac glycosides on senescent or cancer cells suggests the ion pump blockade and intracellular K+ depletion should be synergized with target apoptotic signal to induce the cell death.

Generation of Therapeutically Potent Spheroids from Human Endometrial Mesenchymal Stem/Stromal Cells.

Endometrial mesenchymal stem/stromal cells (eMSCs) hold great promise in bioengineering and regenerative medicine due to their high expansion potential, unique immunosuppressive properties and multilineage differentiation capacity. Usually, eMSCs are maintained and applied as a monolayer culture. Recently, using animal models with endometrial and skin defects, we showed that formation of multicellular aggregates known as spheroids from eMSCs enhances their tissue repair capabilities. In this work, we refined a method of spheroid formation, which makes it possible to obtain well-formed aggregates with a narrow size distribution both at early eMSC passages and after prolonged cultivation. The use of serum-free media allows this method to be used for the production of spheroids for clinical purposes. Wound healing experiments on animals confirmed the high therapeutic potency of the produced eMSC spheroids in comparison to the monolayer eMSC culture.

"All-In-One" Genetic Tool Assessing Endometrial Receptivity for Personalized Screening of Female Sex Steroid Hormones.

Endometrium is the uterine lining that undergoes hundreds of cycles of proliferation, differentiation, and desquamation throughout a woman's reproductive life. Recently, much attention is paid to the appropriate endometrial functioning, as decreased endometrial receptivity is stated to be one of the concerns heavily influencing successes of embryo implantation rates and the efficacy of in vitro fertilization (IVF) treatment. In order to acquire and maintain the desired endometrial receptivity during IVF cycles, luteal phase support by various progestagens or other hormonal combinations is generally recommended. However, today, the selection of the specific hormonal therapy during IVF seems to be empirical, mainly due to a lack of appropriate tools for personalized approach. Here, we designed the genetic tool for patient-specific optimization of hormonal supplementation schemes required for the maintenance of endometrial receptivity during luteal phase. We optimized and characterized in vitro endometrial stromal cell (ESC) decidualization model as the adequate physiological reflection of endometrial sensitivity to steroid hormones. Based on the whole transcriptome RNA sequencing and the corresponding bioinformatics, we proposed that activation of the decidual prolactin (PRL) promoter containing ancient transposons MER20 and MER39 may reflect functioning of the core decidual regulatory network. Furthermore, we cloned the sequence of decidual PRL promoter containing MER20 and part of MER39 into the expression vector to estimate the effectiveness of ESC decidual response and verified sensitivity of the designed system. We additionally confirmed specificity of the generated tool using human diploid fibroblasts and adipose-derived human mesenchymal stem cells. Finally, we demonstrated the possibility to apply our tool for personalized hormone screening by comparing the effects of natural progesterone and three synthetic analogs (medroxyprogesterone 17-acetate, 17α-hydroxyprogesterone caproate, dydrogesterone) on decidualization of six ESC lines obtained from patients planning to undergo the IVF procedure. To sum up, we developed the "all-in-one" genetic tool based on the MER20/MER39 expression cassette that provides the ability to predict the most appropriate hormonal cocktail for endometrial receptivity maintenance specifically and safely for the patient, and thus to define the personal treatment strategy prior to the IVF procedure.

Three-Dimensional Compaction Switches Stress Response Programs and Enhances Therapeutic Efficacy of Endometrial Mesenchymal Stem/Stromal Cells.

Mesenchymal stem cells are currently tested as a promising tool for the treatment of a wide range of human diseases. Enhanced therapeutic potential of spheroids formed from these cells has been proved in numerous studies, however, the fundamental basics of this effect are still being discussed. In this work, we showed that endometrial mesenchymal stem/stromal cells (eMSCs) assembled in spheroids possess a higher therapeutic efficacy compared to cells grown in monolayer in the treatment of the defects that are non-specific for eMSC tissue origin - skin wounds. With the purpose to elucidate the possible causes of superior spheroid potency, we compared the tolerance of eMSC cultivated in spheres and monolayer to the stress insults. Using genetically encoded hydrogen peroxide biosensor HyPer, we showed that three-dimensional configuration (3D) helped to shield the inner cell layers of spheroid from the external H2O2-induced oxidative stress. However, the viability of oxidatively damaged eMSCs in spheroids appeared to be much lower than that of monolayer cells. An extensive analysis, which included administration of heat shock and irradiation stress, revealed that cells in spheroids damaged by stress factors activate the apoptosis program, while in monolayer cells stress-induced premature senescence is developed. We found that basal down-regulation of anti-apoptotic and autophagy-related genes provides the possible molecular basis of the high commitment of eMSCs cultured in 3D to apoptosis. We conclude that predisposition to apoptosis provides the programmed elimination of damaged cells and contributes to the transplant safety of spheroids. In addition, to investigate the role of paracrine secretion in the wound healing potency of spheroids, we exploited the in vitro wound model (scratch assay) and found that culture medium conditioned by eMSC spheroids accelerates the migration of adherent cells. We showed that 3D eMSCs upregulate transcriptional activator, hypoxia-inducible factor (HIF)-1, and secret ten-fold more HIF-1-inducible pro-angiogenic factor VEGF (vascular endothelial growth factor) than monolayer cells. Taken together, these findings indicate that enhanced secretory activity can promote wound healing potential of eMSC spheroids and that cultivation in the 3D cell environment alters eMSC vital programs and therapeutic efficacy.

Proliferation-related changes in K+ content in human mesenchymal stem cells.

Intracellular monovalent ions have been shown to be important for cell proliferation, however, mechanisms through which ions regulate cell proliferation is not well understood. Ion transporters may be implicated in the intracellular signaling: Na+ and Cl- participate in regulation of intracellular pH, transmembrane potential, Ca2+ homeostasis. Recently, it is has been suggested that K+ may be involved in "the pluripotency signaling network". Our study has been focused on the relations between K+ transport and stem cell proliferation. We compared monovalent cation transport in human mesenchymal stem cells (hMSCs) at different passages and at low and high densities of culture as well as during stress-induced cell cycle arrest and revealed a decline in K+ content per cell protein which was associated with accumulation of G1 cells in population and accompanied cell proliferation slowing. It is suggested that cell K+ may be important for successful cell proliferation as the main intracellular ion that participates in regulation of cell volume during cell cycle progression. It is proposed that cell K+ content as related to cell protein is a physiological marker of stem cell proliferation and may be used as an informative test for assessing the functional status of stem cells in vitro.

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

BACKGROUND: Asherman'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. METHODS: Human 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. RESULTS: Application 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. CONCLUSIONS: Human 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.

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.

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 ß-galactosidase (SA-ß-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 72 h 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.