Quiescent Human Mesenchymal Stem Cells Are More Resistant to Heat Stress than Cycling Cells.

Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly, little is known about how quiescent cells respond to environmental challenges. The aim of the present study is to compare stress responses of cycling and quiescent mesenchymal stem cells (MSC). Human endometrial mesenchymal cells (eMSС) were employed as adult stem cells. eMSC quiescence was modeled by serum starvation. Sublethal heat shock (HS) was used as a stress factor. Both quiescent and cycling cells were heated at 45°C for 30 min and then returned to standard culture conditions for their recovery. HS response was monitored by DNA damage response, stress-induced premature senescence (SIPS), cell proliferation activity, and oxidative metabolism. It has been found that quiescent cells repair DNA more rapidly, resume proliferation, and undergo SIPS less than proliferating cells. HS-enforced ROS production in heated cycling cells was accompanied with increased expression of genes regulating redox-active proteins. Quiescent cells exposed to HS did not intensify the ROS production, and genes involved in antioxidant defense were mostly silent. Altogether, the results have shown that quiescent cells are more resistant to heat stress than cycling cells. Next-generation sequencing (NGS) demonstrates that HS-survived cells retain differentiation capacity and do not exhibit signs of spontaneous transformation.

Molecular Genetic Analysis of Human Endometrial Mesenchymal Stem Cells That Survived Sublethal Heat Shock.

High temperature is a critical environmental and personal factor. Although heat shock is a well-studied biological phenomenon, hyperthermia response of stem cells is poorly understood. Previously, we demonstrated that sublethal heat shock induced premature senescence in human endometrial mesenchymal stem cells (eMSC). This study aimed to investigate the fate of eMSC-survived sublethal heat shock (SHS) with special emphasis on their genetic stability and possible malignant transformation using methods of classic and molecular karyotyping, next-generation sequencing, and transcriptome functional analysis. G-banding revealed random chromosome breakages and aneuploidy in the SHS-treated eMSC. Molecular karyotyping found no genomic imbalance in these cells. Gene module and protein interaction network analysis of mRNA sequencing data showed that compared to untreated cells, SHS-survived progeny revealed some difference in gene expression. However, no hallmarks of cancer were found. Our data identified downregulation of oncogenic signaling, upregulation of tumor-suppressing and prosenescence signaling, induction of mismatch, and excision DNA repair. The common feature of heated eMSC is the silence of MYC, AKT1/PKB oncogenes, and hTERT telomerase. Overall, our data indicate that despite genetic instability, SHS-survived eMSC do not undergo transformation. After long-term cultivation, these cells like their unheated counterparts enter replicative senescence and die.

[Long-term cultivation of Chinese hamster fibroblasts of line V-79 RJK under elevated temperature leads to karyotype structure destabilization].

In this article we show that long-term cultivation of Chinese hamster fibroblasts of line V-79 RJK at elevated temperature resulted in the selection of variants with genetic changes at the level of karyotype. From the first steps of resistance selection to elevated temperature we identified population of cells with changes in karyotype (polyploidy cells, deletions, inversions, translocations of chromosomes, and some cells with DM-chromosomes). Further cultivation was accompanied with selection of cells with paracentrical chromosome breakages and HSR's on chromosomes. Nonspecific destabilization of the karyotype (on first steps of selection) was associated with increased expression of hsc70 and pgp. After long-term incubation at an elevated temperature, the cells with karyotypic changes had the basal level of hsc70 and pgp expression.

[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.

[Regulation of proliferation and viability of tumor cells in vitro by alloferon-1 and allostatin-1].

Alloferon-1 (AF) and allostatin-1 (AS) cytotoxic and growth modulating activities have been compared. AF is cationic oligopeptide isolated from the hemolymph of experimentally infected blow fly Calliphora vicina. AS is AF synthetic analog that differs from the parent molecule in two amino acids substituted. It has been shown that both AF and AS have no direct cytotoxic activity in concentrations ranging from 1 x 10(-1) to 10 microg/ml, however, the peptides demonstrated significant effect on tumor cells proliferation in vitro. Both peptides displayed growth modulating activity in mass cell cultures and boosted growth inhibiting activity of doxorubicin in the course of P388D1 cells cloning, although AS potentated doxorubicin cytostatic activity to a greater extent. Similarly, AS boosted anti-clonogenic activity of cyclophosphamide applied in a subthreshold concentration. Experiments with peptide-fluorescein complex have demonstrated that AF and AS belong to the group of cell-penetrating peptides. Moreover, the experiments displayed AF ability to bind with chromosomes.

[The effect of synthetic polycation polyallylamine on adhesion and viability of CHL V-79 RJK Chinese hamster fibroblasts with different heat resistance].

The effects of synthetic polycation polyallylamine (PAA) on adhesion of CHL V-79 RJK fibroblasts and CHL V-79 RJK40 cells resistant to 40 degrees C, and attachment to these cells to polycation immobilized on polystyrene surface were studied. We have also investigated the cytotoxicity of PAA. It was shown that cell adherence to polystyrene plastic coated with PAA was enhanced or decreased in dependence of the PAA concentration used for surface coating and did not depend on heat resistance of investigated cell lines. The effect of PAA on cell adhesion to uncoated polystyrene surface after cell exposure with PAA depended not only on the polycation concentration, but also on the extent of heat resistance of investigated cell lines. Pretreatment of CHL V-79 RJK cells with PAA at the nontoxic concentrations led to inhibition of cell adhesion, and no change in adhesive properties of thermoresistant cells was found under the same conditions. PAA was toxic for CHL V-79 RJK and CHL V-79 RJK40 cells only at concentrations of 100 microg/ml (MTT assay). PAA-induced acute toxicity was accompanied by necrotic-like cell damage. Possible mechanisms of the PAA effect on the behaviour of cells with different structural and metabolic characteristics that are due to the temperature of cell cultivation are discussed.

[Generation of dopamine neurons from human embryonic stem cells in vitro].

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.

[Growth modulating and cytotoxic effects of the peptide from hemolymph of blow fly Calliphora vicina (Diptera, Calliphoridae) in vitro].

Biological activity of alloferon (AF), peptide, consisting of 13 a. a. isolated from hemolymph of experimentally infected blow fly Calliphora vicina was studied. AF in concentrations form 1 x 10(-5) to 250 microg/ml was added into the culture medium of the target cell lines K562, J-96, P388DI, Hep22a and 3T3B-SV40. First two days the peptide in concentrations 1 x 10(-5) and 1 x 10(-3) microg/ml in most cases stimulated the cell proliferative activity and suppressed the cell growth when applied in concentrations 10 and 100 microg/ml. Trend in growth modulating effect was dependent on duration of AF treatment. The peptide did not expressed cytotoxic effect with the exception of destruction of P388D1 cells that was registered after 96 h incubation in the medium initially contained 100 microg/ml AF. Simultaneously, cytotoxic and growth modulating effects of doxorubicin and cytosinarabinoside, as well as hybrid molecules, AF--cytosinarabinoside (cytal) and AF--doxorubicin (doxal) have been studied. Doxorubicin and cytosinarabinoside expressed greater growth inhibition effect compared to the hybrid molecules and AF itself. The results obtained with mass cell cultures were supported by experiments where P388D1 cells clonogenic capacity was tested. Besides, it has been demonstrated that AF rapidly penetrates into cytoplasm of J-96 cells and concentrates mainly into and around the cell nuclei.

[Novel human embryonic stem cell lines C612 and C910].

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.

[Spontaneous transformation and immortalization of mesenchymal stem cells in vitro].

Mesenchymal stem cells (MXC) possess plasticity and unlimited proliferative activity in vitro that makes them an attractive subject of the studies focused on searching new resources for regenerative medicine. The usage of MSC is effective for treatment of patients with degenerative and traumatic diseases of different tissues, however, biological basis of therapeutic efficacy of MSC are still obscure. We found that long term culture of MSC expressing transgenic green fluorescence protein (GFP) led to increase in their proliferative activity, decrease in adhesion, and loss of differentiation potential and production of the GFP. MSC at the first passages showed karyotipic features of transformation, that at the later passages were complicated with developing of tumorigenic abilities detected after transplantation into normal syngenic recipients. When explanted into cell culture conditions the cells of the tumor tissue originated from the MSC did not express GFP and were not inducible to differentiation, but in contrast to the parent cells showed decreased clonogenic and proliferative activities. We suggest that growth of MSC in vitro results in their spontaneous transformation at early passages. Immortalization making physiological basis for the unlimited proliferation of MSC in vitro may be a feature of MSC transformation but not an initial characteristic of the stem cells.

[Effect of ectopic superexperssion of the anti-apoptotic gene bcl-2 on the level and character of karyotypic instability in the CHLL V-79 RJK Chinese hamster cell line]. / Vliianie éktopicheskoi sverkhékspressii antiapoptoticheskogo gena bcl-2 na uroven' i kharakter kariotipicheskoi nestabil'nosti v kletkakh kitaiskogo khomiachka linii CHL V-79 RJK

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.

[Pleiotropic changes in karyotype structure of Chinese hamster fibroblast cell lint CHL V-79 RJK in relation to their acquisition of resistance to etoposide, an inducer of apoptosis]. / Pleiotropnye izmeneniia struktury kariotipa fibroblastov kitaiskogo khomiachka linii CHL V-79 RJK v sviazi s priobreteniem imi ustoichivosti k étopozidu-induktory apoptoza.

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.