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Ionic homeostasis of human mesenchymal stem cells (MSCs) during their long-term cultivation was studied. It was found that, at early passages (second to fourth), the intracellular K+ content calculated for total cellular protein was almost 40% higher than in cells of the same clone after 12–15 passages. Under the same conditions, the intracellular Na+ content remains practically unchanged in MSCs with age. Flow cytometry assay of MSC proliferation at various passages revealed that a decrease in the intracellular K+ content correlated with the cell accumulation in the G1 phase of the cell cycle and was accompanied by a slower cell division. On the basis of data on monovalent ion transport in permanent cell lines of different origin, human stem cells as well as activated human lymphocytes, the mechanism of implication of K+ in the cell proliferation has been discussed. It is suggested that changes in cellular K+ content per total cell protein accompanying triggering or inhibition of cell proliferation show the involvement of K+ in the cell volume regulation. The high intracellular K+ content is important for successful proliferation of hMSCs, and the cell K+ content per cell protein is an informative test for assessing the functional status of stem cells in vitro. [ABSTRACT FROM AUTHOR]

In this study, we reported proliferation-related changes in cell K+ and Na+ content and K+ influxes in cultivated human mesenchymal stem cells (MSCs). The intracellular K+ content calculated per cell protein amount was found to decrease in the growing culture, whereas intracellular Na+ content was not significantly changed. It was also revealed that, at higher densities of an hMSC monolayer, the ouabain-sensitive K+ influx was decreased thus indicating a decline in Na+, K+ pump-mediated transport. We analyzed the cell cycle profiles of hMSC cultures and found that, under optimal culture conditions in high-density cultures, the decline in K+ content per cell protein was correlated with accumulation of G1 cells and slowing down cell proliferation in the population. It is concluded that cell K+ content per cell protein is an informative test for assessing the functional status of stem cells in vitro. [ABSTRACT FROM AUTHOR]

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , 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 H 2 O 2 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 H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 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 H 2 O 2 ., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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 H 2 DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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 β-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 β-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.

Mesenchymal stem cells isolated from human endometrium (eMSC) are perspective source of stem cells for regenerative medicine. Large amount of these cells accumulated by in vitro cultivation is usually required for transplantation into patients. We established several cell eMSC lines and cultivated them during long period of time to examine the possibility of their spontaneous transformation. All cell lines demonstrate limited lifespan, undergo replicative senescence and die. Karyotypic analysis on different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation that makes therapeutic application of these cells safety for patients. During long-term cultivation eMSCs sustain the expression of surface markers.

Oxidative stress has been shown to induce 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 controversial. 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, 350-400 and 600-700 μM for hESC, human embryonic fibroblasts and hMESC, respectively. Thus, among the cell lines studied, hMESC demonstrated the most resistance to increased H2O2 concentration. We have found for the first time that sub-lethal doses of H2O2 induce premature senescence phenotype in hMESC, like in HEF, which is characterized by increased expression of cyclin-dependent kinase inhibitor p21(Waf1/Cip1), an irreversible cell cycle arrest, the permanent loss of proliferative potential, cell hypertrophy and SA-β-Gal staining. While a sub-lethal H2O2 dose (200 μM) promoted in hMESC only SIPS, the higher H2O2 doses induced also apoptosis in the part of the cell population. On the contrary, in hESC, H2O2 regardless of the doses tested (from 50 to 500 μM) triggered apoptosis, that was the only pronounced response of these cells to oxidative damage. The data obtained demonstrate that stem cells of various origins under oxidative stress utilize the different defense mechanisms: hESC rapidly eliminate damaged cells through apoptosis, whereas hMESC may enter SIPS.

Cytotoxic effect of anti-cancer drug, doxorubicin (DR), has been examined on human embryonic stem cells (ESC) C910 and fibroblasts spontaneously differentiated from these cells. The fibroblasts retained diploid karyotype. It was found that ESC are more sensitive to DR than fibroblasts: DR dose killing 20% cells was 0.01 and 0.1 microg/ml, respectively. DR induced ESC apoptotic death and reduced both ESC and fibroblast proliferation. Unlike fibroblasts DR reversibly inhibited ESC proliferation. Thus, we have demonstrated that ESC and their differentiated derivates differ their sensitivity and response to the genotoxic agent.

The aim of the study was to generate dopaminergic (DA) neurons from human embryonic stem cells (ESC) in vitro. It was shown that human ESCs are able to differentiated into DA neurons without co-culture with stromal cells. Terminal differentiation into DA neurons was reached by successive application of noggin and bFGF growth factors on collagen and matrigel substrates during 3-4 weeks. Differentiation efficiency was evaluated by the number of colonies with cells expressing tyrosine hydroxylase (TH), a DA neuron marker, and by the number of TH-positive cells in cell suspension using flow cytometry. No cells with pluripotent markers were detected in DA-differentiated cultures. It makes possible to propose that the protocol of human ESC differentiation might be applied to generate DA neurons for their transplantation into the animals modeling neurodegenerative (Parkinson) disease without the risk of tumor growth.

Novel human embryonal stem cell lines C612 and C910 have been established from hatching blastocytes. Cells were cultivated in mTeST medium on mouse fibroblast feeder-layers. They express common pluripotent markers such as alkaline phosphatase, Oct 3/4, SEEA-4, Nanog, Rex1. Immunophenotyping of these cells by flow cytometry revealed expression of CD90 (Thy-1) and CD117 (c-kit) antigens and weak or no expression of CD13, CD34, CD45, CD130, HLA class I and HLA class II antigens. This pattern of surface antigen expression is common for human embryonic stem cells. G-banding assay of C612 and C910 metaphase plates showed that karyotypic structure of these cells was normal both in chromosome number and structure. The cells are pluripotent because of their capability to generate embryoid bodies, undergo spontaneous differentiation and express markers of all germ layers: nestin, keratin, vimentin (ectoderm), alpha-fetoprotein (entoderm), and muscle alpha-actinin (mesoderm). Thus, C612 and C910 cells have all attributes of typical human embryonic stem cells (diploid, capable of self-renewal, express pluripotent markers and differentiate into three germ layers) and may be of potential use for fundamental and regenerative medicine researches.

The karyotype structure was studied for three cell lines obtained from cells of transgenic murine embryos at early stages of their establishment. The first line was obtained from a transgenic embryonic explantant containing oncogen v-sis under promotor MMTV, two other lines originated from cells of transgenic embryos containing oncogen k51. The karyotypic analysis of G-banded metaphase chromosomes revealed deviations from the normal mouse karyotype as early as by the third passage of cultivation of independent embryonic cell lines that contained a foreign oncogene in their genome. The repeated analysis that involved 15-22 passages revealed similar abnormalities: variability and progression in chromosome number with the appearance of hyperpolyploid combinations, and a large number of rearranged chromosomes, both marker and unique ones. It is concluded that introduction of a foreign oncogene into murine cell genome leads to its enhanced and progressive non-specific destabilization. Oncogen v-sis produces a more valuable karyotype destabilization than oncogen k51.

Karyotypic destabilization in cells of Chinese hamster fibroblasts CHL V-79 RJK with ectopically overexpressed antiapoptotical human bcl-2 gene from pSFFV-bcl-2 vector has been analysed. Analysis of G-banded metaphase chromosomes from 4 clones with different levels of bcl-2 expression revealed an increased level of chromosomal instability in bcl-2-transfected cells. Besides, an increased percentage of aneu- and polyploid cells and high level of cells with different chromosomal aberrations was observed. The degree of karyotypic instability positively correlated with the level of bcl-2 expression in bcl-2-transfected cells. Cells of a clone with the highest bcl-2 expression at the 13th passage of cultivation displayed an almost 100% polyploidization and the presence of specific aberrations and a tricentric marker chromosome. Selection of cells with non-random specific chromosome changes was observed in pSFFV-bcl-2-transfected CHL V-79 RJK cells in the process of their long-term cultivation. By contrast, cells of the parental cell line, as well as the control pSFFV-neo transfectants, displayed a stable karyotype throughout the long period of cultivation. It is important that the presence of morphological markers of gene amplifications--DOO, DM, MH--was observed in bcl-2-transfected cells. These findings suggest that the overexpression of antiapoptotic human bcl-2 gene may result in destabilization of the karyotype structure in cells of Chinese hamster fibroblasts CHL V-79 RJK. The character and level of destabilization correlate with the level of ectopic overexpression of this gene.

Chinese hamster fibroblasts CHL V-79 RJK were subjected to multistep selection in the presence of etoposide, known as an inhibitor of topoisomerase II and inductor of apoptosis. The karyotype of cells stably resistant to etoposide was analysed at progressive stages of selection using G-type staining of metaphase chromosomes. Multiple changes in the karyotype of resistant cells were observed at an early stage of selection (0.2 mg/ml of etoposide) and included: random chromosome breaks leading to formation of new chromosome markers, high frequency of aneuploid and polyploid cells, morphological instability and extracopy of q-shoulder of chromosome 1. On advanced stages of selection we observed an increased frequency of specific minute chromosome and the appearance of chromosomes with homogeneously or differentially stained regions (HSR and DSR). These data suggest that different mechanisms may be involved in developing cellular resistance to etoposide at progressive stages of selection.

A collection of established cell lines was made by means of their explanation into 15 day old transgenic rat embryos. Some of these cell lines were characterized by measuring the cultivated population redoubling time, the saturation density and oncogenicity. A cytogenetic analysis was also carried out. The phenotypical analysis and studies of reproduction permit to define these lines as transformed immortalized non-oncogenic lines capable of contact inhibition. Cytogenetic studies were performed only on Mos N3, N6 and Mos+Neo N1, N6 lines. The karyotypes of cells in Mos lines were normal, and the karyotypes of cells in Mos+Neo lines had chromosomal markers (2 and 3, resp.). These markers result from arrangements of chromosomes 6, 9, 14, 15 and 17. the "thru deletions" of region q1 2qter (line N1) and region q22qter (line N6) of chromosome 15 were revealed in Mos+Neo lines by the summarized reconstruction karyotype method. We propose that these deletions of chromosome 15 and other chromosomes rearrangements may play an important role in transformation of cells from transgenic embryos in vitro, because RB1 antioncogene was mapped on rat chromosome 15.