N. N. Nikolsky

Multipotent mesenchymal stem cells of desquamated endometrium: Isolation, characterization, and application as a feeder layer for maintenance of human embryonic stem cells

In this study, we characterize new multipotent human mesenchymal stem cell lines (MSCs) derived from desquamated (shedding) endometrium of 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 MSCs of any origin. The eMSCs have positive expression of CD13, CD29, CD44, CD73, CD90, and CD105 markers and lack hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130, and HLA-DR (class II). Multipotency of the established eMSCs is confirmed by their ability to differentiate into other mesodermal lineages, such as osteocytes and adipocytes. In addition, the isolated eMSCs partially (over 50%) express the pluripotency marker SSEA-4. However, they do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and β-III-tubulin. This suggests a neural predisposition of the established eMSCs. These cells are characterized by a high proliferation rate (doubling time 22–23 h) and a high colony-forming efficiency (about 60%). In vitro, the eMSCs undergo more than 45 population doublings without karyotypic abnormalities. We demonstrate that mitotically inactivated eMSCs are perfect feeder cells for maintenance of human embryonic stem cell lines (hESCs) C612 and C910. The eMSCs, being a feeder culture, sustain the hESC pluripotent status that verified by expression of Oct-4, alkaline phosphatase and SSEA-4 markers. The hESCs cocultured with the eMSCs retain their morphology and proliferative rate for more than 40 passages and exhibit the capability for spontaneous differentiation into embryoid bodies comprising three embryonic germ layers. Thus, an easy and noninvasive isolation of the eMSCs from 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 hESCs to clinical setting.

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 a substantial number of 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 marker SSEA-4 and neuronal precursors markers nestin and beta-III-tubulin. Further analysis revealed a transcription of the neuronal marker MAP2 and neurotrophin-3 in the undifferentiated BMSCs and ADSCs. Additionally, a significant basal level of synthesis of brain-derived neurotrophic factor (BDNF) in the eMSC culture was also observed. Stimulation of neural induction with agents such 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 the BMSCs proceeded without the RA participation while pretreated with 5-azacytidine. In contrary, in 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 the derived eMSCs appeared to be critical, though an incubation of the cells with 5-azacytidine was optional. As far as the derived 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 is 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 a nerve tissue.

Novel human embryonic stem cell lines C612 and C910

Novel human embryonic stem cell lines C612 and C910 have been established from atching blastocytes. Cells were cultivated in mTeSR medium on a mouse fibroblast feeder layer; they exhibit common pluripotent markers, such as alkaline phosphatase, Oct 3/4, SSEA-4, Nanog, Rex1. The immunophenotyping of these cells by flow cytometry revealed CD90 (Thy-1) and CD117 (c-kit) antigens and showed weak or no expression of CD13, CD34, CD45, CD130, and HLA class I and II antigens, which is typical for human embryonic stem cells. Karyotypic structure of C612 and C910 assayed by the G-banding of metaphase plates is normal in both chromosome number and structure. The cells generate embryoid bodies, undergo spontaneous differentiation, and express three germ-layer markers (nestin, keratin, vimentin ectoderm), α-fetoprotein (entoderm), muscle α-actinin (mesoderm), i.e., possess pluripotent potency. Thus, C612 and C910 display accepted human embryonic stem cell properties, including unlimited self-renewal, expression of pluripotent markers, ability to differentiate into three germ layers, and are diploid; therefore, they may be of potential use for fundamental research, as well as for replacement therapy studies.

Somatic polyploidy is associated with the upregulation of c-MYC interacting genes and EMT-like signature

The dependence of cancer on overexpressed c-MYC and its predisposition for polyploidy represents a double puzzle. We address this conundrum by cross-species transcription analysis of c-MYC interacting genes in polyploid vs. diploid tissues and cells, including human vs. mouse heart, mouse vs. human liver and purified 4n vs. 2n mouse decidua cells. Gene-by-gene transcriptome comparison and principal component analysis indicated that c-MYC interactants are significantly overrepresented among ploidy-associated genes. Protein interaction networks and gene module analysis revealed that the most upregulated genes relate to growth, stress response, proliferation, stemness and unicellularity, as well as to the pathways of cancer supported by MAPK and RAS coordinated pathways. A surprising feature was the up-regulation of epithelial-mesenchymal transition (EMT) modules embodied by the N-cadherin pathway and EMT regulators from SNAIL and TWIST families. Metabolic pathway analysis also revealed the EMT-linked features, such as global proteome remodeling, oxidative stress, DNA repair and Warburg-like energy metabolism. Genes associated with apoptosis, immunity, energy demand and tumour suppression were mostly down-regulated. Noteworthy, despite the association between polyploidy and ample features of cancer, polyploidy does not trigger it. Possibly it occurs because normal polyploidy does not go that far in embryonalisation and linked genome destabilisation. In general, the analysis of polyploid transcriptome explained the evolutionary relation of c-MYC and polyploidy to cancer.

Mesenchymal stem cells from human endometrium do not undergo spontaneous transformation during long-term cultivation

Human-endometrium mesenchymal stem cells (eMSCs) are a promising source of stem cells for regenerative medicine. A large amount of these cells accumulated by in vitro cultivation are usually required for transplantation into patients. We established several cell eMSC lines and cultivated them over a long period to examine the possibility of spontaneous transformation. All cell lines exhibit limited lifespan, undergo replicative senescence, and die. Karyotypic analysis upon different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation, which makes therapeutic application of these cells safe for patients. During long-term cultivation, eMSCs maintain the expression of surface markers.

Induction of decidual differentiation in endometrial mesenchymal stem cells

In this study, we compared the ability of human mesenchymal stem cells (eMSCs) derived from menstrual blood and mesenchymal stem cells (MSCs) from other tissues to differentiate into decidual cells in vitro. It was demonstrated that, during differentiation, secretion of prolactin and insulin-like growth factor binding protein-1 (key decidualization markers) markedly increased in eMSCs slightly augmented in bone marrow MSC (BM-MSCs) and did not change in MSCs from adipose tissue (AT-MSCs). Thus, eMSCs exhibited higher capacity for differentiation into decidual cells than BM-MSCs or AT-MSCs. This makes eMSCs promising for application in cellular therapy of infertility associated with insufficient decidualization of endometrium.

Brain-derived neurotrofic factor (BDNF) secretion of 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 the MSCs from different donors, isolated from the bone marrow (BMSC), subcutaneous adipose tissue (AD MSC) and menstrual blood (eMSC). It was established that the native eMCSs, BMSCs and AD MSCs express neuronal marker β-III-tubulin with a frequency of 90, 50 and 14%, respectively. Also we showed that the 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. An induction of neuronal differentiation in the studied MSCs using differentiation medium containing B27 and N2 supplements, 5-azacytidine, retinoic acid, IBMX and dbcAMP induced changes in the cells morphology, the increase of β-III-tubulin expression, and the appearance of neuronal markers GFAP, NF-H, NeuN and MAP2. During the differentiation the BDNF secretion 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 in the studied MSCs has been established.

Different protective mechanisms of human embryonic and endometrium-derived mesenchymal stem cells under oxidative stress

Oxidative stress has been shown to cause either apoptosis or stress-induced premature senescence (SIPS) in different cell types. At present, it is generally accepted that stem cells have high resistance to oxidative stress; however, data reported by various authors are disputed. In this study, we investigated stress responses of human embryonic stem cells (hESC) and human mesenchymal stem cells (hMESC) derived from desquamated endometrium to hydrogen peroxide (H2O2). Cell viability was evaluated by MTT assay. LD50 were determined as 300–350, 370–400, and 600–700 μM for hESC, human embryonic fibroblasts, and hMESC, respectively. Thus, of the studied cell lines, hMESC exhibited the greatest resistance to increased H2O2 concentration. We found for the first time that a sublethal concentration of H2O2 induced premature senescence phenotype in hMESC, like in HEF, that was characterized by increased expression of cyclin-dependent kinase inhibitor p21Waf1/Cip1, an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy, and the SA-β-Gal staining. Whereas the sublethal H2O2 concentration (200 μM) promoted in hMESC only SIPS, higher H2O2 concentrations also induced apoptosis in a small part of the cell population. On the contrary, in hESC, H2O2, regardless of the tested concentrations (from 50 to 500 μM), triggered apoptosis, which was the only pronounced response of these cells to oxidative damage. The obtained data demonstrate that stem cells of different origins under conditions of oxidative stress use different protective mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC are subjected to premature senescence.

[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 the p53/p21/Rb and p38/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However, the interrelations between the p53/p21/Rb and MAPK signaling pathways, including ERK1/2, p38, and JNK, remain yet unexplored. 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 of the proteins. Each MAPK suppression significantly increased the p53 phosphorylation level, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation rather than p38 and JNK. These results suggest the existence of a reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. By analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as ERK antagonists: JNK is able to activate ERK, while p38 may block ERK activation. Together, these results demonstrate the existence of complex links between different signaling cascades in stressed hMESC, implicating ERK, p38, and JNK in regulation of premature senescence via the p53/p21/Rb pathway.

Production and characterization of monoclonal antibodies against human endoglin

Endoglin (CD105), a component of TGF-β, BMP-9, BMP-10 binding receptor complexes, is the marker of endothelial and mesenchymal stem cells. Its expression is significantly increased in the blood vessel endothelium of ischemic tissues and growing tumors. Measurement of the endoglin soluble form in serum and urine is used in diagnostics of cancer and pregnancy disorders. The aim of this work was to produce a panel of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. A recombinant protein derived from murine myeloma cells representing the whole extracellular part of endoglin was used as the immunogen. F1(SJL/J × BALB/c) mice were the donors of immune splenocytes. Hybridoma screening was performed using E. coli-produced recombinant molecules, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight different epitopes were produced. Eight antibodies bind the membrane-associated 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 to measure soluble endoglin in the cell culture medium.