G. G. Poljanskaya

Russian Academy of Sciences, Moskva, Moscow, Russia

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Publications (8)0.98 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: We developed a feeder-free system for human embryonic stem cells (ESCs) based on extracellular matrix protein (ECM) as the substrate. ECM was synthesized by mesenchymal stem cells (SC5-MSC) derived from an original ESC line, SC5. The ECM proteins fibronectin and laminin facilitate ESC growth in the feeder-free system. An important component of this system is a conditioned medium from SC5-MSC cells. Two ESC sublines were obtained: SC5-FF cells were cultured in an autogenic, and SC7-FF in an allogenic, feeder-free system. SC5-FF and SC7-FF underwent more than 300 and 115 population doublings, respectively, and retain a normal diploid karyotype. Histochemical and immunofluorescence assays showed that both sublines express undifferentiated ESC markers—alkaline phosphatase, Oct-4, SSEA-4, and TRA-1-81—as well as multidrug resistance transporter ABCG2. PCR assay revealed that undifferentiated SC5-FF cells, like the original SC5 line, maintained on feeder cells express OCT4 and NANOG genes common for somatic cells and DPPA3/STELLA and DAZL genes common for germ line cells. Expression of these genes was gradually diminished during differentiation of embryoid bodies, whereas expression of genes specific for early differentiated cells increased: GATA4, AFP (extraembryonic and embryonic endoderm), PAX6 (neuroectoderm), and BRY (mesoderm). ESC properties (karyotype structure, average time of population doubling, undifferentiated cell number in population) of the SC5 and SC7 and SC5-FF and SC7-FF sublines derived from original ESCs were not altered. It shows that the feeder-free systems, which are more stable than any feeder systems, maintain key ESC properties and may be recommended for fundamental, biomedical, and pharmacological studies performed with human ESCs.
    Cell and Tissue Biology 01/2013; 7(1).
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    ABSTRACT: 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.
    Russian Journal of Developmental Biology 01/2011; 42(4):5-18. · 0.49 Impact Factor
  • G. G. Poljanskaya, T. N. Efremova
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    ABSTRACT: The effect of Mycoplasma salivarium on the numerical and structural karyotypic variability was studied in the markerless cell line of Indian muntjac skin fibroblasts (line M) during long-term cultivation with and without L-arginine. The cultivation of mycoplasma-contaminated cells for 15 and 30 days did not change the character of cell distribution for the number of chromosomes. In contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number changed. These changes involve bimodal distribution for the chromosome number due to a significant decrease in frequency of the cells with the modal number of chromosomes with the main structural variant of karyotype (SVK) 2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with the submodal number of chromosomes with a main SVK of 2 + 2 + 1 + 1. Furthermore, a significant increase in the frequency of cells with lower numbers of chromosomes was observed after 60 days compared to that after 75 days of cultivation. After the cultivation of the contaminated and control cells in the medium with an elevated concentration of L-arginine for 60 days, the numerical parameters were unchanged relative to the control. The cultivation of contaminated cells for 60 days followed by the addition of L-arginine for 15 days restored the numerical parameters to the control level. In the contaminated cells, the frequency of chromosomal aberrations for 30, 60, and 75 days increased significantly compared to the control variant. After 30 days of cultivation, a small but statistically significant increase took place due to a uniform slight increase in the frequency of chromosomal aberrations of all types. After 60 and 75 days, a greater increase occurred due to a statistically significant increase in the frequency of chromosomal and chromatid breaks. Moreover, after 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as for a means of adaptation for markerless cell lines to the condition of cultivation and the role of L-arginine in the restoration of the normal karyotypic structure of the line M cell population at mycoplasmal contamination are discussed. Keywordskaryotypic variability–mycoplasmal contamination–L-arginine–chromosomal aberrations–dicentrics
    Cell and Tissue Biology 01/2011; 5(1):54-61.
  • G. G. Poljanskaya, T. S. Goryachaya, G. P. Pinaev
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    ABSTRACT: Numerical and structural karyotypic variability was investigated in the NBL-3-17 and NBL-3-11 “markerless” rat kangaroo kidney cell lines cultivated on a fibronectin-coated surface. For the NBL-3-17 cell line grown on a fibronectin-coated surface for periods of 1, 2, 4 and 8 days, the chromosome number distribution changed. These changes involved a significant decrease in the frequency of cells with the modal chromosome number and an increase in the frequency of cells with a lower chromosome number. Many new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to the predominant adhesion of cells with a lower chromosome number, disturbances of the mitotic apparatus and selection for SVK adapted to the changes in culture conditions. Detachment of cells from the fibronectin-coated surface followed by 5 days cultivation on a hydrophilic surface restored the control cell distribution. For the NBL-3-11 cell line cultured on the fibronectin-coated surface for 1, 2, 4 and 8 days, the numerical karyotypic variability did not change compared to control variants. For the NBL-3-17 cell line grown on a fibronectin-coated surface for 1, 2, 4 and 8 days, the frequency of chromosomal aberrations also did not change relatively to the control. In the NBL-3-11 cell line, the frequency of chromosomal aberrations under the same conditions significantly increased, mainly due to chromosome and chromatid breaks and dicentrics (telomeric associations). The differences in the numerical and structural karyotypic variability between NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) cell lines cultivated on fibronectin are discussed. It is assumed that the observed differences in the karyotypic variability between these cell lines were determined by the specific karyotypic structure of the NBL-3-11 cell line and the altered gene expression of the NBL-3-17 hypotriploid cell line caused by increased doses of certain functioning genes.
    Cell and Tissue Biology 01/2007; 1(2):115-124.
  • G. G. Poljanskaya, T. S. Goryachaya, G. P. Pinaev
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    ABSTRACT: The numerical and structural karyotypic variability has been investigated in MTs of the markerless cell line of Indian muntjac skin fibroblasts, as well as in its karyotypic variant MTD cultivated on a laminin 2/4 coated surface. In the MT cell line preincubated in serum-free medium for 2.5 and 1.0 h, then cultivated on a laminin-coated surface in serum-containing medium for one, two, and three days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in the frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Some new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to disturbances of the chromosome segregation and the establishment of a new advantageous balanced karyotypic structure. In the karyotypic variant MTD differing from MT by an increased number of dicentrics (telomeric associations) cultivated under the same conditions, the character of cell distribution for the chromosome number did not change. In the MT cell line, the frequency of chromosomal aberrations did not change relative to control variants. In the karyotypic variant MTD under the same conditions, the frequency of chromosomal aberrations significantly increased after three days mainly due to the formation of dicentrics. These results confirm the conclusion that, like aneuploidy, the formation of dicentrics in markerless cell lines appears to be the way in which the cell population adapts to unfavorable environmental factors. Possible reasons for differences in the character of the numerical and structural karyotypic variability between the MT cell line and its karyotypic variant MTD are discussed.
    Cell and Tissue Biology 2(6):625-635.
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    ABSTRACT: We have studied the application of histones for the modification of a cell-culture surface. Experiments were conducted on 293 cell lines (human embryonic kidney cells transformed by Ad5 adenovirus) and BALB/3T3 clone A31 (spontaneously transformed mouse embryonic fibroblasts). The cell’s interactions with various types of histons placed on a hydrophobic glass surface or 1.0 μm dextran microspheres was assessed. It was found that all histones studied exhibited adhesive properties but their complexes, such as the total and core histones, were the most adhesive and had improved cell morphology. Cross-linked conjugates of histone complexes immobilized on microshperes facilitated cell network formation by cellular interactions and cell contacts with several microspheres. For the 293 line, unlike BALB/3T3 clone, the A31 cells significantly increased their proliferative activity on microspheres coated with cross-linked conjugates of histone complexes. The study showed that the substrate may be applied for 3D pore matrices designed for tissue-like cell structures in vitro. Key wordshistones-microspheres-adhesion-cell proliferation in culture
    Cell and Tissue Biology 4(1):14-24.
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    ABSTRACT: Four continuous human embryonic stem cell (ESC) lines (SC1, SC2, SC3, and SC4) isolated from preimplantational blastocysts obtained by artificial fertilization have been described. The cell lines were cultivated on mitotically inactivated human feeder cells. SC1 and SC2 cell lines passed through 150 population doublings, while the SC3 and SC4 cell lines passed through about 120 population doublings, which significantly exceeded Hayflick’s limit. All of these cell lines maintain the high activity of alkaline phosphatase and the expression of transcription factor OCT-4 and cell surface antigens (SSEA-4, TRA-1-60, TRA-1-81), which confirms the status of ESC and human specificity. An immunofluorescent analysis of the expression of antigens that characterize the ectoderm, endoderm, and mesoderm has confirmed the capacity of these cells for pluripotency in all four lines under in vitro conditions. Using PCR analysis, the expression of six genes specific to pluripotent cells, i.e., OCT-4, NANOG, DPPA3/STELLA, TDGF/CRIPTO, and LEFTYA, has been revealed. The correlation between the level of proliferative activity and the character of staining with DNA-bound fluorescent dyes has been found. Hoechst 33342 and PI dyes produced a diffuse staining of nuclei in slowly proliferating cells of the SC1 and SC2 lines. On the contrary, in actively proliferating cells of the SC3 and SC4 lines, a distinct staining of nuclei was observed. Upon changing the cultivation conditions, proliferative activity of the SC3 and SC4 lines decreased and the character of the fluorescent staining became similar to that of the SC1 and SC2 lines. The quality of the fluorescent staining with Hoechst 33342 and propidium iodide reflects the level of proliferation. Possible causes and mechanisms of this peculiarity of human ESCs are discussed. Key wordscontinuous human embryonic stem cell lines-immunofluorescent analysis-expression of genes-antibodies-fluorescent dyes
    Cell and Tissue Biology 4(1):1-13.
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    ABSTRACT: 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.
    Russian Journal of Developmental Biology 44(1). · 0.49 Impact Factor