[Expression of Cancer-Testis Antigens of Magea and Mageb Families in Mouse Embryonic Fibroblasts Cultured in vitro].

Cancer-testis antigens are expressed in the spermatogenic and cancer cells as well as in human and mouse pluripotent stem cells. However, the role of cancer-testis antigens of Mage families in the regulation of cellular processes in embryonic cells is largely unknown. In the present study comparative quantitative analysis of the gene expression of Magea and Mageb families was performed in mouse embryonic somatic cells (mouse embryonic fibroblasts, MEFs), long-term cultured in vitro and exposed to factors that inhibit and stimulate proliferation. The analysis revealed low expression of cancer-testis antigens of Mage families and showed that a lower proliferative activity of MEF at late passages was accompanied by slight up-regulation of the Magea gene expression and down-regutation of Mageb gene expression. However, modulation of the activity of MEK/ERK-signaling pathway and DNA demethylation by 5-azacytidine had no significant effects on the expression of Magea and Mageb genes in M EFs. The most essential changes in the expression levels of Mageb and Magea genes were found only when MEFs were exposed to mitomycin C. In all experimental variants, predominantly cytoplasmic localization of Mage antigens was found in MEFs at the phase of DNA synthesis, as well as at other cell cycle phases. Presumably, in actively proliferating mouse embryonic somatic cells the antigens of Magea and Mageb families can act as co-activators in the regulation of cell proliferation and other cellular functions.

[Low expression of activin A in mouse and human embryonic teratocarcinoma cells].

TGFP3 family factors play an important role in regulating the balance of self-renewal and differentiation of mouse and human pluripotent stem and embryonic teratocarcinoma cells. The expression patterns of TGFbeta family signaling ligands and functional roles of these signaling pathways differ significantly in mouse and human embryonic stem cells, but the activity and functional role of these factors in mouse and human embryonic teratocarcinoma cells were not sufficiently investigated. Comparative quantitative real-time PCR analysis of the expression of TGF@[beta] family factors in mouse embryonic stem, embryonic germ, and embryonic teratocarcinoma cells showed that embryonic teratocarcinoma cells express lower ActivinA than pluripotent stem cells but similar levels of factors Nodal, Lefty 1, TGFbeta1, BMP4, and GDF3. In human nullipotent embryonic teratocarcinoma PA-1 cells, most factors of the TGFbeta family (ACTIVINA, NODAL, LEFTY 1, BMP4, and GDF3) are expressed at lower levels than in human embryonic stem cells: Thus, in mouse and human nullipotent teratocarcinoma cells, theexpression of ActivinA is significantly reduced com- pared ivith embryonic stem cells. Presumably, these differences may be associated with changes in the functional activity of the respective signaling pathways and deregulation of proliferative and antiproliferative mechanisms in embryonic teratocarcinoma cells.

[Expression of TGFbeta family factors and FGF2 in mouse and human embryonic stem cells maintained in different culture systems].

Mouse and human embryonic stem cells are in different states of pluripotency (naive/ground and primed states). Mechanisms of signaling regulation in cells with ground and primed states of pluripotency are considerably different. In order to understand the contribution of endogenous and exogenous factors in the maintenance of a metastable state of the cells in different phases ofpluripotency, we examined the expression of TGFbeta family factors (ActivinA, Nodal, Leftyl, TGFbeta1, GDF3, BMP4) and FGF2 initiating the appropriate signaling pathways in mouse and human embryonic stem cells (mESCs, hESCs) and supporting feeder cells. Quantitative real-time PCR analysis of gene expression showed that the expression patterns of endogenous factors studied were considerably different in mESCs and hESCs. The most significant differences were found in the levels of endogenous expression of TGFbeta1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFbeta1, and FGF2 for hESCs are feeder cells (mouse and human embryonic fibroblasts) expressing high levels of these factors, as well as low levels of BMP4. Thus, our data demonstrated that the in vitro maintenance of metastable state of undifferentiated pluripotent cells is achieved in mESCs and hESCs using different schemes of the regulations of ActivinA/Nodal/Lefty/Smad2/3BMP/Smad1/5/8 endogenous branches of TGFbeta signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFbeta family factors and FGF2 in the mESCs and hESCs. For the hESCs, enhanced activity of ActivinA/Nodal/Lefty/Smad2/3 signaling by exogenous factor stimulation is necessary to mitigate the effects of BMP/Smadl/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primary states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.

[Cytotoxic effects of etoposide at different stages of differentiation of embryoid bodies formed by mouse embryonic stem cells].

The initial stages of in vitro differentiation of embryonic stem cells are considered as unique three-dimensional models of early development of mammals for basic, pharmacological, and toxicological studies. It has been previously shown (Gordeeva, 2012) that the assessment of embryotoxicity in the model of undifferentiated embryonic stem cells can be insufficiently accurate in predicting toxic effects on mammalian embryos. In view of this, we performed a comparative study of the damaging effects of the cytostatic etoposide in undifferentiated embryonic stem cells and embryoid bodiesof different stages of differentiation that have similar three-dimensional structures with early embryos. The analysis of growth, cell death, and dynamics of differentiation of embryonic stem cells and embryoid bodies exposed to etoposide showed that the cytostatic and cytotoxic effects of etoposide are stage-specific. The damaging effects of etoposide were maximum in the undifferentiated embryonic stem cells and decreased with growth and differentiation of embryoid bodies. We assume that the increase in the cell volume of embryoid bodies and the development of the hypertrophic we suggest that the increase of embryoid body volume and overgrowth of extraembryonic endoderm layer lead to a decrease in the diffusion, transport, and metabolism of chemical and bioactive substances and prevent the damaging effects.

Development of experimental tumors formed by mouse and human embryonic stem and teratocarcinoma cells after subcutaneous and intraperitoneal transplantations into immunodeficient and immunocompetent mice.

Pluripotent stem cells represent an attractive cell source for regenerative medicine. However, the risk of teratoma formation after transplantation restricts their clinical application. Therefore, to adequately evaluate the potential risk of tumorigenicity after cell transplantation into human tissues, effective animal transplantation assays need to be developed. We performed a multiparameter (cell number, transplantation site, cell type, host) comparative analysis of the efficiency of tumor development after transplantation of mouse and human embryonic stem (ES) cells and their malignant counterparts, teratocarcinoma (EC) cells, into animal recipients and revealed several key correlations. We found that the efficiency of tumor growth was higher after intraperitoneal than after subcutaneous transplantations of all cell lines studied. The minimal cell numbers sufficient for tumor growth in immunodeficient nude mice were 100-fold lower for intraperitoneal than for subcutaneous transplantations of mouse and human ES cells (10(3) vs. 10(5) and 10(4) vs. 10(6), respectively). Moreover, mouse ES and EC cells formed tumors in immunodeficient and immunocompetent mice more effectively than human ES and EC cells. After intraperitoneal transplantation of 10(3), 10(4), and 10(5) mouse ES cells, teratomas developed in 83%, 100%, and 100% of nude mice, whereas after human ES cell transplantation, teratomas developed in 0%, 17%, and 60%, respectively. In addition, malignant mouse and human EC cells initiated tumor growth after intraperitoneal transplantation significantly faster and more effectively than ES cells. Mouse and human ES cells formed different types of teratomas containing derivatives of three germ layers but different numbers of undifferentiated cells. ES cell-like sublines with differentiation potential similar to the parental cell line were recloned only from mouse, but not from human, ES cell teratomas. These findings provide new information about the possibility and efficiency of tumor growth after transplantation of pluripotent stem cells. This information allows one to predict and possibly prevent the possible risks of tumorigenicity that could arise from stem cell therapeutics.

[Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells].

Pluripotent stem cells are able to proliferate indefinitely and differentiate in vitro into various cell types. However, in most cases in vitro differentiation of the pluripotent stem cells is asynchronous and incomplete, and the residual undifferentiated cells can initiate teratoma development after transplantation into recipients. These features of the pluripotent stem cells are the major issue for development of safe cell therapy technologies based on pluripotent stem cells. Considering significant resemblance of growth rates of pluripotent stem and cancer cells we investigated antiproliferative and cytotoxic effects of different type cytostatics (mitomycin C, etoposide, vinblastine and cycloheximide) on the undifferentiated and differentiating mouse embryonic stem cells, embryonic germ cells, blastocyst and on mouse embryonal teratocarcinoma cells and mouse embryonic fibroblasts. The findings showed that all cytostatics used induced both antiproliferative effects and acute toxic processes in undifferentiated pluripotent stem cells and embryonal teratocarcinoma cells whereas these effects were less in differentiating embryonic stem cells and embryonic fibroblast. Moreover, the trophoblast cells of mouse blastocysts were less sensitive to damaging effects of cytostatics than inner cell mass cells. The examination of deferred effects ofcytostatics revealed that the effects ofmitomycin C, etoposide and vinblastine, but not cycloheximide, were irreversible because survived cells were not able to proliferate. Nevertheless, the numbers of embryonic fibroblasts exposed to etoposide or vinblastine remained unchanged while vast majority of undifferentiated pluripotent cells treated underwent apoptosis. Thus, diverse effects of etoposide and vinblastine on the undifferentiated pluripotent stem cells and differentiated embryonic cells allow us to consider these cytostatics and their analogs as drug-candidates for selective elimination of the residual undifferentiated pluripotent stem cells from population of differentiating cells. These findings demonstrate for the first time the possibility of selective elimination of undifferentiated pluripotent stem cells using cytostatic drugs approved for clinic practice. However, to improve effectiveness and safety of this approach and to prevent mutagenic, carcinogenic and teratogenic effects on undifferentiated pluripotent stem cells and their differentiated cell derivatives large-scale studies ofcytostatic effects using different experimental design and active doses must be performed.

[Developing of a new feeder-free system and characterization of human embryonic stem cell sublines derived in this system under autogenic and allogenic culturing].

A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells--mesenchymal stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins--fibronectin and laminin--that maintain hESC growth in feeder-free system were identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatase, Oct-4, SSEA-4, TRA-1-81 and multidrug resistance transporter--ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA ans DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP (extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features (karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs.

[Comparative characteristics of new human embryonic stem cell lines SC5, SC6, SC7, and SC3a].

Numerous human embryonic stem cell lines with different genetic background are widely used as cell models for fundamental, biomedical and pharmacological research. New hES cell lines SC5, SC6, SC7, and SC3a are derived from the blastocysts and maintained on mitotically inactivated human feeder cells. All derived hES cell lines passed through more than 120 cell population doublings, retained normal diploid karyotype and ability of in vitro differentiation in the derivates of three germ layers. These lines express the markers of undifferentiated hES cells: Oct-4, Nanog, SSEA-4, TRA-1-60, and alkaline phosphatase. Moreover, undifferentiated cells of SC5, SC6, and SC7 lines expressed germ line specific genes DPPA3/STELLA and DAZL and did not express somatic lineages specific genes. In contrast, undifferentiated cells of SC3a line did not express DPPA3/STELLA and DAZL but expressed extra embryonic endoderm cell markers GATA4 and AFP. Double staining of SC5 and SC3a colonies by antibodies against transcription factors Oct-4 and GATA4 has demonstrated that most SC3a cells in colonies were positive for both factors. Furthermore, the cells of SC5, SC6, SC7 lines but not of SC3a line formed teratomas containing the derivates of the three germ layers. These results indicate that, in contrast to the other cell lines, the cells in the SC3a colonies represent an early committed cell population. Moreover, expression of the multidrug resistance transporter gene ABCG2 was detected in undifferentiated cells and differentiating embryonic bodies during 10 days of all lines by immunofluorescent and RT-PCR analyses, whereas RT-PCR analysis has revealed up-regulation of the ABCB1 transporter gene expression in differentiating embryoid bodies of SC5, SC6, and SC7 cells only. Thus, these findings demonstrate different characteristics and differentiation potential of SC5, SC6, SC7, and SC3a hES cell lines which were derived in different conditions.

Pluripotent cells in embryogenesis and in teratoma formation.

Pluripotent cells of the early preimplantation embryo originate all types of somatic cell and germ cells of the adult organism. Permanent pluripotent cell lines (ES and EG cells) that were derived from an inner cell mass of blastocysts and primordial germ cells have a high proliferative potential and ability to differentiate in vitro into a wide variety of somatic and extraembryonic tissues as well as germ cells and to contribute to different organs of chimeric animals. In some cases pluripotent cells and primordial germ cells can generate teratomas, teratocarsinomas and some kinds of seminomas as the results of damages of differentiation programme of these cells. Experimental teratomas which formed after transplantation of undifferentiated ES and EG cells into immunocompromiced mice may provide a unique opportunity to study pluripotent cell specification and to develop novel approaches in carcinogenesis investigations. Research of signaling and metabolic pathways regulating the pluripotent cell maintenance and their multilineage differentiation are essential to search molecular targets to eliminate undifferentiated cells in tumors. Analysis of interactions between pluripotent cells and differentiated cells of the recipient animals, identification of the factors that may drive differentiation ES and EG cells in vivo contribute in understanding the mechanisms involved in the determination of cell fate during normal development and tumorigenesis. These data are important for development of effective and safe stem cell based technologies for prospective clinical treatment.

[The characters and specific features of new human embryonic stem cells lines].

Four continuous human embryonic stem cell lines (SC1, SC2, SC3 and SC4), derived from the blastocysts has been described. The cell lines were cultivated on mitotically inactivated human feeder cells. The cell lines SC1 and SC2 have passed through 150 population doublings and the cell lines SC3 and SC4 -- near 120 populations doublings, which exceeds Hayflick limit sufficiently. These cell lines maintain high activity of alkaline phosphatase, expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60 and TRA-1-81), confirming their ESC status and human specificity. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm confirms the ability of these cells to retain their pluripotency under in vitro condition. PCR analysis revealed expression of six genes specific for pluripotent cells (OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO and LEFTYA). Correlation between the level of proliferative activity and the character of DNA-bound fluorescent staining was found. Fluorescent dyes, Hoechst 33342 and PI, produced diffuse staining of the nuclei in slowly proliferating cells of the SC1 and SC2 lines. In contrast, in actively proliferating cells of the SC3 and SC4 lines, the clear staining of the nuclei was observed. Upon changing the cultivation condition, proliferative activity of SC3 and SC4 lines decreased and became similar to that of SC1 and SC2 lines. The character of the fluorescent staining of all these lines was also shown to be similar. These results show that quality of the fluorescent staining with Hoechst 33342 and PI reflects the level of proliferation. Possible causes and mechanisms of this feature of human ESC are discussed.

[Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells, and teratocarcinoma cells by TGFb family signaling factors].

The activity of specific signaling and transcription factors determines the cell fate in normal development and in tumor transformation. The transcriptional profiles of gene-components of different branches of TGFbeta family signaling pathways were studied in experimental models of initial stages of three-dimensional in vitro differentiation of embryonic stem cells, embryonic germ cells and teratocarcinoma cells and in teratomas and teratocarcinomas developed after their transplantation into immunodeficient Nude mice. Gene profile analysis of studied cell systems have revealed that expression patterns of ActivinA, Nodal, Lefty1, Lefty2, TGF TGFbeta1, BMP4, and GDF were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of Inhibin betaA/ActivinA were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFbeta family factors regulates pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFbeta family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

[Pluripotent stem cells: maintenance of genetic and epigenetic stability and prospects of cell technologies].

Permanent lines of pluripotent stem cells can be obtained from humans and monkeys using different techniques and from different sources--inner cell mass of the blastocyst, primary germ cells, parthenogenetic oocytes, and mature spermatogonia--as well as by transgenic modification of various adult somatic cells. Despite different origin, all pluripotent lines demonstrate considerable similarity of the major biological properties: active self-renewal and differentiation into various somatic and germ cells in vitro and in vivo, similar gene expression profiles, and similar cell cycle structure. Ten years of intense studies on the stability of different human and monkey embryonic stem cells demonstrated that, irrespective of their origin, long-term in vitro cultures lead to the accumulation of chromosomal and gene mutations as well as epigenetic changes that can cause oncogenic transformation of cells. This review summarizes the research data on the genetic and epigenetic stability of different lines of pluripotent stem cells after long-term in vitro culture. These data were used to analyze possible factors of the genome and epigenome instability in pluripotent lines. The prospects of using pluripotent stem cells of different origin in cell therapy and pharmacological studies were considered.

[Comparative analysis of expression of TGFbeta family factors and their receptors in mouse embryonic stem and embryonic teratocarcinoma cells].

Specific factors that determine the cell fate in early embryogenesis are modulated during interaction of signaling pathways to form a unique regulatory network inside the cell, which is essential for differentiation of various cell populations. We carried out a comparative study of expression of the genes of TGFbeta growth factors and their receptors at the initial stages of differentiation of the embryonic stem cells, during formation of spheroids of the embryonic teratocarcinoma cells, and during growth of neoplastic cells in vivo in immunodeficient mice. The patterns of expression of the genes Activin, Nodal, Lefty1, Lefty2, BMP, and TGF1 and their receptors ActRI, ActRII, BMPRI, TGFbeta1R1, and Tdgf proved to be identical. Expression of alpha-fetoprotein and transcription factor Gata4 protein, specific for the primary endoderm, was detected in the embryonic teratocarcinoma cells. In Undifferentiated embryonic stem cells, expression of Gata4 was found at the mRNA level, while expression at the level of proteins appeared only in the primary endoderm cells in the embryoid bodies. The results obtained suggest that despite the existence of similar signaling systems in the embryonic stem and teratocarcinoma cells, the presence of different intracellular specific factors forms radically different regulatory pathways, which determine the program of their differentiation.

[Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro]. / Ekspressiia reguliatornykh genov Oct-4, Pax-6, Prox-1, Ptx-2 na nachal'nykh stadiiakh differentsirovki émbrional'nykh stvolovykh kletok in vitro.

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4 involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

[Differentiation of pluripotent embryonic stem cells in peritoneal cavity of irradiated mice]. / Izuchenie differentsirovki pliuripotentnykh émbrional'nykh stvolovykh kletok v peritoneal'noi polosti obluchennykh myshei.

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies grafted in abdominal cavity of the irradiated mice were shown to form tumors with derivatives of all germinal layers. Cells of the embryoid bodies form tumors two weeks after the grafting, while the undifferentiated embryonic stem cells form tumors only by week three.

[A biological model of accelerated aging. III. Histological characteristics of age-related changes of spermatogenic epithelium in mice SAM (senescence-accelerated mouse)]. / Biologicheskaia model' uskorennogo stareniia. III. Gistologicheskaia kharakteristika vozrastnykh izmenenii spermatogennogo épiteliia u myshei linii SAM (senescence-accelerated mouse).

The data characterizing the age-related morphological changes in the spermatogenic epithelium of SAMP1 (senescence-accelerated prone) and SDAMR1 (senescence-accelerated resistant) mice are presented. In many tubules, "early spermatogenesis" was accompanied by the formation of many morphologically abnormal germ cells on histological sections of the gonads of sexually immature (three-four weeks) mice of both strains. At this stage, destructive processes in the spermatogenic epithelium were more pronounced in SAMR1 mice. In sexually mature (two-three months) SAMP1 and SAMR1 mice, spermatogenesis as a whole proceeded normally. The first signs of regressive changes in the inner structure of most tubules (disintegration, detachment of spermatogenic epithelium from basal membrane) and morphology of germ cells (pycnosis, nuclear and cytoplasmic vacuolization) were found in SAMP1 mice at the age of six-seven months. In the older age groups (9-10 and 12-15 months), all types of spermatogenic cells were represented in both SAMP1 and SAMR1 mice, but most of these cells were atypical. Mitotic figures were recorded in a population of highly differentiated Sertoli cells.

[Biological model of accelerated senescence. II. Age-related changes in the number of developing male germ cells and Sertoli cells in gonads of senescence-accelerated mice]. / Biologicheskaia model' uskorennogo stareniia. II. Vozrastzavisimye izmeneniia chisla razvivaiushchikhsia muzhskikh polovykh kletok i kletok Sertoli v gonadakh myshei linii SAM (senescence-accelerated mouse).

It was shown that in immature three- to four-week-old mice prone to accelerated senescence (SAMP1 strain), the number of spermatogonia, pachytene spermatocytes, and circular spermatids exceeded that in mice resistant to accelerated senescence (SAMR1 strain) by more than two times. Differences were found in the pattern of age-related changes in the number of meiotic and postmeiotic cells in the sexually mature SAMP1 and SAMR1 mice. In the gonads of SAMP1 and SAMR1 mice, the number of Sertoli cells was unstable.