O. F. Gordeeva

Comparative analysis of expression of TGFβ family factors and their receptors in mouse embryonic stem and 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 TGFβ 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 TGFβ1 and their receptors ActRI, ActRII, BMPRI, TGFβ1R1, and Tdgf proved to be identical. Expression of α-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.

Regulation of in vitro and in vivo differentiation of mouse embryonic stem cells, embryonic germ cells and teratocarcinoma cells by TGFβ 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 TGFβ 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, Tgfβ1, Bmp4, and GDF3 were identical in pluripotent stem cells whereas the mRNAs of all examined genes with the exception of (Inhibin Inhibin βA/ActivinA) were detected in the teratocarcinoma cells. These results indicate that differential activity of signaling pathways of the TGFβ family factors regulates the pluripotent state maintenance and pluripotent stem cell differentiation into the progenitors of three germ layers and extraembryonic structures and that normal expression pattern of TGFβ family factors is rearranged in embryonic teratocarcinoma cells during tumor growth in vitro and in vivo.

[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 for 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 lineage specific genes. In contrast, undifferentiated cells of the 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, and SC7 lines but not of the SC3a line formed teratomas containing 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 differentiated embryonic bodies (EB) of all lines during 10 days by immunofluorescent and RT-PCR analysis, 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 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.

Expression of Regulatory GenesOct-4, Pax-6, Prox-1, and Ptx-2 at the Initial Stages of Differentiation of Embryonic Stem Cells 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-4involved 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.

Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E-ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ cells as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency.

Analysis of expression of genes specific for pluripotent and primordial germ cells in human and mouse embryonic stem cell lines.

115 Permanent lines of embryonic stem cells (ESCs) of humans and animals are promising cell models for fundamental, biomedical, pharmacological, and toxicological studies. Today, the world collection of human ESC lines numbers approximately 300 lines, including the reconstructed lines obtained using the technology of somatic cell nucleus transfer into the enucleated oocyte and the lines carrying genetic lesions [1–4]. Human ESC lines obtained at Russian institutes (Institute of Cytology and Institute of Gene Biology, Russian Academy of Sciences) were characterized earlier using the standard tests detecting the expression of the embryonic surface antigens SSEA3 and SSEA4, the transcription factors OCT4 and NANOG, as well as the activity of alkaline phosphatase and telomerase [4].

Expression of cancer-testis antigens of Mage-a and Mage-b 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, a comparative quantitative analysis of the Mage-a and Mage-b gene expression was performed in mouse embryonic somatic cells (mouse embryonic fibroblasts, MEFs) long-term cultured in vitro or exposed to factors that inhibit and stimulate proliferation. The analysis revealed a low expression of cancer-testis antigens of Mage families and showed that the decrease in proliferative activity of MEFs at late passages was accompanied by slight up-regulation of the Mage-a gene expression and down-regulation of Mage-b gene expression. However, modulation of the MEK/ERK-signaling pathway activity and DNA demethylation with 5-azacytidine had no significant effects on the Mage-a and Mage-b gene expression in MEFs. The most essential changes in the expression levels of Mage-a and Mage-b genes were found only when MEFs were exposed to mitomycin C. In all experimental variants, the predominant cytoplasmic localization of Mage antigens was found in MEFs at the DNA synthesis stage, as well as at other stages of the cell cycle. Presumably, in actively proliferating mouse embryonic somatic cells, the antigens of Mage-a and Mage-b families can act as coactivators in the regulation of cell proliferation and other cellular processes.

Expression of TGFβ 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 of pluripotency, we examined the expression of TGFβ family factors (ActivinA, Nodal, Lefty1, TGFβ1, 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 TGFβ1, BMP4 and ActivinA. The sources of exogenous factors ActivnA, TGFβ1, 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/Smad2/3 and BMP/Smad1/5/8 endogenous branches of TGFβ signaling. The requirement for exogenous stimulation or inhibition of these signaling pathways is due to different patterns of endogenous expression of TGFβ 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/Smad1/5/8 signaling pathways that promote cell differentiation into the extraembryonic structures. Significant differences in endogenous FGF2 expression in the cells in the ground and primed states of pluripotency demonstrate diverse involvement of this factor in the regulation of the pluripotent cell self-renewal.

Expression of cancer-testis antigens of the Mage family in mouse oocytes and early embryos

Cancer-testis antigens of the Mage family (Melanoma antigens) are expressed predominantly in the spermatogenic and cancer cells, but some genes of this family are expressed ubiquitously. Expression patterns and functional role of Mage family antigens in the regulation of cellular processes in normal embryonic and definitive cells are virtually unknown. Comparative immunofluorescent analysis of Mage expression in mouse oocytes and early embryos identified the expression of Mage antigens at all stages studied. The greatest intensity of the fluorescent staining was detected in the epiblasts and the extraembryonic structures of the egg cylinder at E6.5 stage. At all studied developmental stages of the mouse oocyte and the early embryo, the localization of Mage antigens was found predominantly in the cytoplasm. Quantitative real-time PCR showed that expression levels of most Mage genes in cells of the epiblast and ectoplacental cone were similar, while the gene expression levels of Mage-a10, Mage-b16, and Mage-b18 were higher in cells of the ectoplacental cone than in epiblast cells. Thus, for the first time, our analysis has shown that the Mage family antigens are expressed at the early stages of mouse development and may be involved in the regulation of earliest events of embryogenesis.