Mesenchymal stem cells lose the senescent phenotype under 3D cultivation.

BACKGROUND: Three-dimensional (3D) cell culture is widely used in various fields of cell biology. In comparison to conventional two-dimensional (2D) cell culture, 3D cell culture facilitates a more accurate replication of the in vivo microenvironment, which is essential for obtaining more relevant results. The application of 3D cell culture techniques in regenerative medicine, particularly in mesenchymal stem cell (MSC)-based research, has been extensively studied. Many of these studies focus on the enhanced paracrine activity of MSCs cultured in 3D environments. However, few focus on the cellular processes that occur during 3D cultivation. METHODS: In this work, we studied the changes occurring within 3D-cultured MSCs (3D-MSCs). Specifically, we examined the expression of numerous senescent-associated markers, the actin cytoskeleton structure, the architecture of the Golgi apparatus and the localization of mTOR, one of the main positive regulators of replicative senescence. In addition, we assessed whether the selective elimination of senescent cells occurs upon 3D culturing by using cell sorting based on autofluorescence. RESULTS: Our findings indicate that 3D-MSCs were able to lose replicative senescence markers under 3D cell culture conditions. We observed changes in actin cytoskeleton structure, Golgi apparatus architecture and revealed that 3D cultivation leads to the nuclear localization of mTOR, resulting in a decrease in its active cytoplasmic form. Additionally, our findings provide evidence that 3D cell culture promotes the phenotypic reversion of senescent cell phenotype rather than their removal from the bulk population. CONCLUSION: These novel insights into the biology of 3D-MSCs can be applied to research in regenerative medicine to overcome replicative senescence and MSC heterogeneity as they often pose significant concerns regarding safety and effectiveness for therapeutic purposes.

[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.

[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.

[Effect of bone marrow cells transplantation on the decidua formation in pseudopregnant rats].

One of the most common causes of the current pregnancy loss is the failure of the decidual reaction of endometrial cells. It is assumed that a partial source of decidual cells in endometrial tissue is bone marrow cells (BMCs). In the present work, we have studied possible effect of BMCs transplantation on the process of decidualization using the model of pseudopregnancy in rats. BMCs were flushed from the rat femurs and tibias. The obtained suspension of single BMCs was injected into one of rat uterine horns on the 5th day of pseudopregnancy. PBS without cells was injected into the contralateral horn served as the control. Rats were sacrificed on the 11th day of pseudopregnancy. Decidua formed in the experimental uterine horn showed an increase in the meso-antimezometral direction of their diameter of about 1.5-2 times as compared with a control horn. The weight of decidual tissue in the experimental horn exceeded 3 times the weight of the control one. The presence of transplanted BMCs in decidual tissue was documented by preliminary double staining of BMCs with membrane dye PKH 26 Red and nuclear dye Hoechst 33342. Histological analysis of decidua sections after transplantation revealed any alterations neither in cell differentiation nor in tissue structure. We conclude that BMCs transplantation stimulates decidualization in animals.

[BDNF secretion in human mesenchymal stem cells isolated from bone marrow, endometrium and adipose tissue].

The ability of mesenchymal stem cells (MSCs) to differentiate into neuronal lineage determines the potential of these cells as a substrate for a cell replacement therapy. In this paper we compare the neurogenic potential of MSCs isolated from bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was found that the native eMCSs, BMSCs and AD MSCs express neuronal marker ß-III-tubulin with a frequency of 90, 50 and 14%, respectively. We also showned that eMSCs have a high endogenous level of brain-derived neurotrophic factor (BDNF), whereas the BMSCs and the AD MSCs are characterized by low basal BDNF levels. As induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMF caused changes in the cells morphology, the increased expression of ß-III-tubulin, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. BDNF secretion during differentiation was significantly enhanced in the BMSCs and decreased in the eMSCs cultures. However, no correlation between the basal and induced levels of the neuronal markers expression and BDNF secretion in the studied MSCs has been established.

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.

[Neurogenic potential of human mesenchymal stem cells isolated from bone marrow, adipose tissue and endometrium: a comparative study].

Mesenchymal stem cells (MSCs) can be isolated from many adult tissue sources. These cells are a valuable substrate in cell therapy for many diseases and injuries. Different types of MSCs vary in plasticity. We performed a comparative study of the neurogenic potential of three types of human MSCs derived from bone marrow (BMSCs), subcutaneous adipose tissue (ADSCs) and endometrium (isolated from the menstrual blood) (eMSCs). It was shown that all three types of MSC cultures demonstrate multipotent plasticity and predisposition to neurogenesis, based on the expression of pluripotency markers SSEA-4 and neuronal precursors' markers nestin and beta-III-tubulin. Further analysis revealed the transcription of the neuronal marker MAP2 and neurotrophin-3 in undifferentiated BMSCs and ADSCs. Additionally, a significant basal level of synthesis of brain-derived neurotrophic factor (BDNF) in eMSC culture was also observed. Stimulation of neural induction with such agents as 5-azacytidine, recombinant human basic fibroblast growth factor (bFGF), recombinant human epidermal growth factor (EGF), a recombinant human fibroblast growth factor 8 (FGF8), morphogen SHH (sonic hedgehog), retinoic acid (RA) and isobutyl-methyl-xanthine (IBMX), showed further differences in the neurogenic potential of the MSCs. The components of the extracellular matrix, such as Matrigel and laminin, were also the important inducers of differentiation. The most effective neural induction in BMSCs proceeded without the RA participation while the cells pretreated with 5-azacytidine. In contrary, in the case of eMSCs RA was a necessary agent of neural differentiation as it stimulated the transcription of neurotrophin-4 and the elevation of secretion level of BDNF. The use of laminin as the substrate in eMSCs appeared to be critical, though an incubation of the cells with 5-azacytidine was optional. As far as ADSCs, RA in combination with 5-azacytidine caused the elevation of expression of MAP2, but reduced the secretion of BDNF. Thus, the effect of RA on neural differentiation of ADSCs in ambiguous and, together with the study of its signaling pathways in the MSCs, requires further research. The therapeutic effect of transplanted MSCs is commonly explained by their paracrine activity. The high basal level of BDNF synthesis in the eMSCs, along with their high proliferative rate, non-invasive extraction and neural predisposition, is a powerful argument for the use of the intact eMSCs as a substrate in cell therapy to repair nerve tissue.

[Multipotent mesenchymal stem cells of desquamated endometrium: isolation, characterization and use as feeder layer for maintenance of human embryonic stem cell lines].

In this study, we characterize new multipotent human mesenchymal stem cell (MSC) lines derived from desquamated (shedding) endometrium in menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSC of any origin. The eMSCs have positive expression of CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II). Multipotency of the established eMSC is confirmed by their ability to differentiate into other mesodermal cell types such as osteocytes and adipocytes. Besides, the isolated eMSC lines partially (over 50%) express the pluripotency marker SSEA-4, but do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and beta-III-tubulin. This suggests a neural predisposition of the established eMSC. These cells are characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). In vitro the eMSCs undergo more than 45 population doublings revealing normal karyotype without karyotipic abnormalilies. We demonstrate, that the mititotically inactivated eMSCs are perfect feeder cells for human embryonic stem cell lines (hESC) C612 and C910. The eMSC being a feeder culture maintain the pluripotent status of the hESC, which is revealed by the expression of Oct-4, alkaline phosphatase and SSEA-4. When co-culturing, hESC retain their morphology, proliferative rate for more than 40 passages and capability for spontaneous differentiation into embryoid bodies comprising the three embryonic germ layers. Thus, an easy and non-invasive extraction of the eMSC in menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESC to clinical setting.