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.

Impact of Polyallylamine Hydrochloride on Gene Expression and Karyotypic Stability of Multidrug Resistant Transformed Cells.

The synthetic polymer, polyallylamine hydrochloride (PAA), is found in a variety of applications in biotechnology and medicine. It is used in gene and siRNA transfer, to form microcapsules for targeted drug delivery to damaged and tumor cells. Conventional chemotherapy often does not kill all cancer cells and leads to multidrug resistance (MDR). Until recently, studies of the effects of PAA on cells have mainly focused on their morphological and genetic characteristics immediately or several hours after exposure to the polymer. The properties of the cell progeny which survived the sublethal effects of PAA and resumed their proliferation, were not monitored. The present study demonstrated that treatment of immortalized Chinese hamster cells CHLV-79 RJK sensitive (RJK) and resistant (RJKEB) to ethidium bromide (EB) with cytotoxic doses of PAA, selected cells with increased karyotypic instability, were accompanied by changes in the expression of p53 genes c-fos, topo2-α, hsp90, hsc70. These changes did not contribute to the progression of MDR, accompanied by the increased sensitivity of these cells to the toxic effects of doxorubicin (DOX). Our results showed that PAA does not increase the oncogenic potential of immortalized cells and confirmed that it can be used for intracellular drug delivery for anticancer therapy.

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.

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.

Senescence as a general cellular response to stress: A mini-review.

Cellular senescence was initially described as the phenomenon of limited cell divisions that normal cells in culture can undergo during long-term-cultivation. Later it was found that senescence may be induced by various stress factors. The intriguing resemblance between stress-induced and replicative senescence makes questionable the distinction between both types and suggests that the cellular senescence is a common outcome of stress response. Growing evidences support the idea that stress-induced senescence is the cell-type specific.

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.

Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny.

Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.