[Comparative characteristics of mesenchymal stem cell lines derived from bone marrow and muscle of limb of early human embryo].

In this work, we have carried out a comparative analysis of the characteristics of mesenchymal stem cell lines isolated from different tissues of 5-6-weeks homan embryo: bone marrow (line FetMSC) and muscle of limb (line M-FetMSC). The basic characteristics of these lines were obtained at the 6th passage. Average population doubling time was 33.0 ± 1.4 h (FetMSC) and 25.0 ± 0.1 h (M-FetMSC). Growth curves also indicated active proliferation of cells of both lines. Numerical and structural karyotypic analysis showed that both lines have a normal karyotype: 46, XY. In order to determine the status of the lines, cell surface markers were analyzed by flow cytometry. The analysis revealed the presence of surface antigens specific for human MSCs, CD44, CD73, CD90, CD105, HLA-ABC, vimentin, and the lack of CD34 and HLA-DR, in both lines. The ability to differentiate into osteogenic, chondrogenic and adipogenic directions has been also shown for both lines. Im- munofluorescence and flow cytometry analysis has detected no expression of the surface antigen TRA-1-60 in both lines, but has revealed high expression of the surface antigen SSEA-4 and low expression of transcription factor Oct-4 characteristic of human embryonic stem cells. In these lines, immunofluorescence analysis has shown the presence of the markers of early differentiation in the derivates of three germ layers characteristic of human embryonic stem cells, which provides significant opportunities for MSC to be useful, in corresponding microenvironments, for repair of tissue injures. Dispite confirming MSC status for FetMSC and M-FetMSC lines, a number of interlinear differences related to growth characteristics and differentiation potential were revealed. Adipogenic differentiatiation potential of M-FetMSC line was reduced compared with FetMSC line. Immunofluorescence analysis showed that, in the process of skeletal-muscle differentiation, Z-disks were revealed only in sarcomeres of M-FetMSC line. These findings suggest the possible influence of different microenvironments in which the cells are in the body before their transfer in vitro.

[Influence of human fetal mesenchymal stem cells on glioma cell proliferation. A consequence of cellular crosstalk].

The effect of mesenchymal stem cells (MSCs) on the growth of various tumors is ambiguous. MSCs derived from different tissues stimulate growth of some tumor types and exert antitumor effects on the other. Several recent reports have shown that crosstalk between tumor cells and MSCs contribute to these effects. The aim of this work was to study the effects of MSCs derived from fetal tissues on the proliferative activity of glioma cells in conditions of prolonged co-cultivation. We have analyzed the proliferative activity of glioma cells exposed to conditioned medium (CM) from MSCs derived from fetal bone marrow (FetMSC) and fetal muscle (M-FetMSC) as well as to CM from co-cultivation of the fetal MSCs with U251MG glioma cells. As a comparison, the influence of CM from adult dermal fibroblasts (DFs) was examined in identical experiments. Using MTT assay, we have found that CM from both the fetal MSCs and adult DFs (without their co-culturing with glioma cells) had no effect on U251MG and A172 glioma cell proliferation. However, CM from early co-cultures (3-9 days) of U251MG cells with FetMSC or M-FetMSC exerted stimulatory effect on U251MG cell proliferation up to 2.3-fold increase, while CM obtained later from the same co-cultures (15-21 days) had inhibitory effect on the proliferation up to arrest of cell division. Analogous experiments with adult DFs have revealed a persistent stimulation of U251MG cell proliferative activity for all 21 days of co-culturing. Immunofluorescence analysis revealed a reduction in the expression of cell cycle protein cyclin D1 in U251MG cells after their treatment with CM taken from 21-days co-cultures of U251MG cells with FetMSC or M-FetMSC. In contrast, CM from 21-days co-cultures of U251MG cells with DFs did not decrease the expression of cyclin D1. These results show that fetal MSCs have dual effect on glioma cell proliferation. In spite of the earlier stimulatory effect on the proliferative activity, prominent inhibition of glioma cell proliferation was observed after three week co-culturing of glioma cells with fetal MSCs. This is the first report demonstrating reversion of tumor cell proliferative program during co-culturing with MSCs for a long time. These data suggest that CM obtained at different time points of cell co-culturing can be used in the modeling of prolonged cellular crosstalk.

[The problem of genomic instability of cultivated human stem cells].

The wide application of human stem cells in biomedical technologies leads to the necessity to analyze the genomic stability of cultivated stem cells of different origin. The review presents data on genetically stable and unstable continuous lines of human embryonic stem cells (hESC), their differentiated derivatives and adult stem cells. Causes of genomic instability occurrence are considered and analysis of the methods used to study the genome of cell lines has been carried out. The results presented in this review demonstrate the need for periodic control of genomic instability of all types of stem cells. Molecular-genetic analysis in order to avoid contamination of adult stem cell lines by tumor cells is also needed. This aspect is particularly important in connection with the active use of the adult stem cells for regenerative medicine.

[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.

[The influence of substrate from extracellular matrix proteins on karyotypic variability of the Indian muntjac skin fibroblast two cell lines].

The effect of cell culture conditions on numerical and structural karyotypic variability was investigated in two Indian muntjac skin fibroblast "markerless" cell lines, M and MT. The cells cultivated on the substrate consisting of extracellular matrix proteins (ECM), synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for chromosome number of cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on hydrophilic surface without ECM-coating. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. MT cell line, differing from M line in the number of homologous chromosomes, demonstrated similar with M line the character of cell distribution for chromosome number only for 1 day after cultivating on the ECM-substrate, but not after 4 days in the same culture conditions, no difference from the control cells was observed. The observed alterations seem to be due to disturbances in correct chromosome segregation process, which were caused by abrupt shift in the cell culture conditions. The analysis of the structural karyotypic variability revealed significant increase in frequency of chromosomal aberrations in M cell line for 1 and 4 days in culture on the ECM-substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured in the same conditions. The obtained results show that the cell lines of the same origin but of different karyotypic structure react to substrate in a different way. In contrast to M line, in MT line a fast normalization of numerical karyotypic characteristics and no enhancement of structural karyotypic variability takes place. This provides a possibility to cultivate MT cell on the given protein substrate maintaining a balanced karyotypic structure characteristic of MT cell line.

[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 mesenchymal stem cell lines derived from human embryonic stem cells, bone marrow and foreskin].

New nonimmortalized fibroblast-like cell lines SC5-MSC and SC3a-MSC, FetMSC, FRSN were obtained from human embryonic stem cells (ESC), bone marrow of a 5-6-days embryo and foreskin of a 3-years-old boy, respectively. All the lines are successfully used as the feeder at human ESC cultivation. It is determined that the average cell population doublings time varies from 25.5 h for ISC5-MSC to 38.8 h for SC3a-MSC. Active proliferation of all the lines is also shown by the corresponding growth curves. Numerical and structural karyotypic analysis showed that these lines had normal karyotype: 46,XX (SC5-MSC and SC3a-MSC) and 46,XY (FetMSC and FRSN). To determine the status of the lines, their cell surface markers were analyzed by flow cytometry. This analysis revealed the presence of surface antigens CD44, CD73, CD90, CD105 and HLA-ABC, characteristic of human MSC, and the absence of CD34 and HLA-DR. Different lines were found to express CD117(c-kit) to a different level. Immunofluorescence and flow cytometry analysis did not detect TRA-1-60 and Oct-4, characteristic of human embryonic stem cells, and revealed interlinear variations in the level of SSEA, which did not depend on the cell origin. It is not clear yet whether these interlinear variations affect functional MSC status. In all the lines, immunofluorescence analysis showed the presence of the markers of early differentiation in the derivates of three germ layers which may allow MSC to be useful, in corresponding microenvironments, for reparation of tissue injures. Adipogenic and osteogenic differentiatiation of all cell lines has been shown.

[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.

[Cultivation of cells on a surface covered by microspheres with coupled histones].

The use of histones for modification of the surface intended for cultivation of cells was studied. The work was carried out on the cell line 293 of human embryonic kidney transformed by adenovirus (Ad5) and on the cell line BALB/3T3 clone A31 of mouse spontaneous transformed embryonic fibroblasts. We analyzed interaction of cells with histones of different types put on a hydrophobic surface or on dextran microspheres with diameters of 1.0 microm. It was shown, that all histones studied possessed adhesive ability, but their complexes consisting of total and core histones rendered the best influence on adhesion, morphology and growth of the cells in culture. Thus, cross-linked conjugates of histones immobilized on microspheres promoted in a greater degree formation of a network of cellular structures due to formation of intracellular contacts and simultaneous interaction of cells with several microspheres. Comparing with BALB/3T3 clone A31 the cell line 293 showed significant increase in proliferative activity in 11 days of cultivation on microspheres covered with cross-linked conjugates of histones. Our investigations have shown that the microspheres covered with cross-linked conjugates of histones can be used in the further at creation of the three-dimensional porous matrices intended for in vitro formation of tissue-like cellular structures in them.

[The influence of Mycoplasma salivarium in the absence and presence of L-arginine on karyotypic variability in cell line of the Indian muntjak skin fibroblasts under long-term cultivation].

The influence of Mycoplasma salivarium on the numerical and structural karyotypic variability has been investigated in the "markerless" cell line of the Indian muntjak skin fibroblasts (line M) during long-term cultivation in the absence and presence of L-arginine. Cultivation of the mycoplasmal contaminated cells for 15 and 30 days did not change the character of cell distribution for the chromosome number. In the contaminated cells cultivated for 60 and 75 days, the character of cell distribution for the chromosome number was changed. These changes involved bimodal distribution for the chromosome number due to a significant decrease in the frequency of the cells with the modal number of chromosomes with main structural variant of karyotype (SVK)--2 + 2 + 1 + 1 + 1 and an increase in the frequency of cells with submodal number of chromosomes with main SVK--2 + 2 + 1 + 1. Besides, a significant increase in the frequency of the cells with lower chromosome number was observed in 60 days compared to that in 75 days of cultivation. Cultivation of the contaminated and control cells in the medium with increased concentration of L-arginine during 60 days did not change the numerical parameters relative to the control. Cultivation of the contaminated cells for 60 days followed by addition of L-arginine for 15 days restored the numerical parameters the numerical parameters to the control level. In the contaminated cells the frequency of chromosomal aberrations significantly increased for 30, 60 and 75 days cultivation relative to the control variant. In 30 days, the small but significant increase took place due to increase in the frequency of chromosomal aberrations of all the types. In 60 and 75 days, a greater increase took place due to a significant increase in the frequency of chromosomal and chromatid breaks. Moreover, in 60 days, the level of dicentrics (telomeric associations) mainly produced by chromosomes 1 and 2 increased significantly. The role of dicentrics as one of the ways for adaptation of the "markerless" cell lines to condition of cultivation and the role of L-arginine in the restoration of normal karyotypic structure of cell population of line M under mycoplasmal contamination are discussed.

[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.

[The influence of immobilized laminin on karyotypic variability in two karyotypic different variants of the Indian muntjac skin fibroblast cell line].

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts "markerless" cell line MT and in its karyotypic variant MT(d) cultivated on the laminin 2/4 coated surface. In cell line MT preincubated in serum free medium for 2.5 and 1 h and then cultivated on the laminin-coated surface in the serum-containing medium for 1, 2 and 3 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Some new additional structural variants of the karyotype (SVK) appeared. The observed alterations seem to be due to disturbances of chromosome segregation and establishing a new advantageous balance karyotypic structure. The karyotypic variant MT(d) distinguished from MT by the increased number of dicentrics (telomeric associations) and cultivated under the same conditions showed no change in the character of cell distribution for the chromosome number. In cell line MT, the frequency of chromosomal aberrations did not change relative to control variants. In karyotypic variant MT(d) under the same conditions. the frequency of chromosomal aberrations significantly increases in 3 days mainly due to formation of dicentrics. These results confirm the conclusion that, similarly to aneuploidy, formation of dicentrics in "markerless" cell lines appears to be the way for cell population to adapt to unfavourable factors of the environment. Possible reasons for differences in the character of the numerical and structural karyotypic variability between cell line MT and its karyotypic variant MT(d) are discussed.

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

[Continuous human embryonic stem cell lines].

A new continuous human embryonic stem cell line (HESC-5) derived from a blastocyst is described. The cultured cell passed over 200 population doublings, which exceeds the Hayflick's limit sufficiently. The cells maintained a stable proliferative activity, high activity of alkaline phosphatase, and expression of transcription factor Oct-4 and of surface antigens SSEA-3, SSEA-4 and TRA-1-60 known to be characteristic of embryonic stem cells of the human origin. Immunofluorescent detection of antigens, characteristic of ectoderm, endoderm and mesoderm in the new cell line HESC-5, and in the previously described other four stem cell lines confirms the ability of these cells to retain their pluripotency under in vitro condition. In addition, in all the cell lines, a high telomerase activity was revealed, which controls a stable telomere length and, hence, an unlimited ESC proliferation. Unlike other cell lines, HESC-5 was found, under specific conditions, to spontaneously differentiate into hematopoietic cells. A morphological similarity was shown between ESC colonies cultivated both on a feeder layer and in the non-feeder system.

[The effect of immobilized fibronectin on karyotypic variability in the skin fibroblast cell subline of indian muntjac].

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblast cell subline MT on cultivating cells on the fibronectin-coated surface. In cell subline MT, cultivated on the fibronectin-coated surface for 1 and 2 days, the character of cell distribution for the chromosome number did not change. In 3, 4 and 8 days, the character of cell distribution for the chromosome number changed. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population. Detachment of cells from the fibronectin-coated surface, followed by a 1 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for the chromosome number, but cultivation in these conditions for 5 days restore control distribution. The frequency of chromosomal aberrations on cultivation on the fibronectin-coated surface for 3 and 4 days significantly increases, mainly at the expence of dicentrics (telomeric association). On prolongating the time of cultivation up to 8 days on the fibronectin-coated surface the frequency of chromosomal aberrations approaches the control value. Structural instability of chromosomes at cultivation on the fibronectin-coated surface demonstrates nonspecific reaction of "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability in cell lines of the Indian muntjac skin fibroblasts on cultivating on laminin and fibronectin.

[Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines]. / Vliianie laminina na kolichestvennuiu kariotipicheskuiu izmenchivost' v kletochnykh liniiakh pochki kengurovoi krysy.

The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous chromosomes (hypotriploid and hypodiploid).

[Effect of laminin on structural karyotype variability of kangaroo rat kidney cell lines]. / Vliianie laminina na strukturnuiu kariotipicheskuiu izmenchivnost' v kletochnykh liniiakh pochki kengurovoi krysy.

The structural karyotypic variability has been investigated in the "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, and in cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, there is a significant increase in the frequency of chromosomal aberrations, both chromosomal breaks and dicentrics (telomeric associations). Different sensitivity of individual chromosomes to inducing chromosomal breaks was observed in addition to a preferential involvement of some chromosomes in dicentric formation. Structural instability of chromosomes at cultivation on laminin demonstrates nonspecific reaction of the "markerless" cell lines to unfavourable factors of the environment. We discuss possible reasons of differences in the character of karyotypic variability between a cell line of the Indian muntjac skin fibroblasts and epithelial-like Rat kangaroo kidney cell lines cultivated on laminin.

[Isolation and characterisation of continuous human embryonic stem cell lines]. / Poluchenie i kharakteristika postoiannykh linii émbrional'nykh stvolovykh kletok cheloveka.

A long-term cultivation (5-8 months) of human blastocyst-derived embryonic cells (hES) was performed. Several properties of hESs were examined to prove the state of continuous cell lines. These cells have passed through 100-175 population doublings with the average population doubling time equal to 37.0 +/- 1.5 h. Isolated hESs, referred to as HESC-1, HESC-2, HESC-3, HESC-4, cultivated on mitotically inactivated mouse embryonic fibroblasts (STO continuous cell line), formed multilayer colonies of various shape. The cells maintained stable proliferative activity, high activity of alkaline phosphatase and expression of transcription factor Oct4, and all this characterizes embryonic stem cells of different origin. Expression of hES specific cell surface antigens (SSEA-3, SSEA-4, TRA-1-81 and TRA-11-81 and TRA-1-60) was confirmed by immunofluorescence analysis with the corresponding monoclonal antibodies. An additional prove for species specificity of HESC lines is the lack of expression of mouse specific surface antigen SSEA1. The cell cycle of HESC-1 undifferentiated cells and embryoid bodies was analysed cytofluorimetrically.

[Effect of laminin on karyotypic variability in cell line of Indian muntjac skin fibroblasts]. / Vliianie laminina na kariotipicheskuiu izmenchivost' v kletochnoi linii fibroblastov kozhi indiiskogo muntzhaka.

The numerical and structural karyotypic variability has been investigated in the Indian muntjac skin fibroblasts cell line M and karyotypic variant of this line M' on cultivation on a laminin 2/4 coated surface. In cell line M, cultivated on the laminin-coated surface for 4 and 14 days, and in karyotypic variant M', cultivated for 2, 4 and 14 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with model numbers of chromosomes, and an increase in frequency of cells with lower chromosome numbers. As a result, new modal chromosome numbers form. The frequency of cells with 4 chromosomes increases significantly; as a rule, such cells are absent in the control cell variants. Many new additional structural variants of the karyotype (SVK) appear. Detachment of cells M' from the laminin-coated surface followed by a 2 day cultivation on a hydrophilic surface, commonly used for routine cell cultivation, does not restore the control cell distribution for chromosomal number. The frequency of chromosomal aberrations on cultivation of the laminin-coated surface does not change relatively to controls. The observed alterations seem to be due to both disturbances of mitotic apparatus and selection of SVK, which are more advantageous to changed culture conditions of the cell population.