[CHARACTERISTICS OF NEW MESENCHYMAL STEM CELL LINE DERIVED FROM HUMAN EMBRYONIC STEM CELLS].

New nonimmortalized fibroblast-like cell line SC6-MSC has been obtained from a line of human embryonic stem cells (ESC)--SC6. Numerical and structural karyotypic analysis has shown hypodiploidy karyotypic: 45, X0 in this line. The average cell population doublings time, for SC6-MSC is 26.0 ± 0.4 h at the 8th passage and 82.0 ± 9.2 h at the 18th passage. The growth curves showed active proliferation for 8-10 passages with a consequent gradual decrease of proliferative activity, which ended to 20th passage. To determine the line's status, the analysis of the surface markers by flow cytometry was carried out. We have revealed the expression of surface antigens CD44, CD73, CD90, CD105 and HLA-ABC characteristic for human MSC, and the absence of CD34 and HLA-DR expression. However, the level of expression of surface markers CD90 and CD105 was significantly lower in comparison with other MSC lines including the line SC5-MSC derived from the line human ESC-SC5. Immunofluorescence analysis of the expression of the surface markers and transcription factor Oct-4 characteristic for human embryonic stem cells showed the absence of Oct-4 expression and the presence of SSEA-4 and TRA-1-60 expression, which is characteristic for a number of MSC lines with normal karyotype. Immunofluorescence analysis has shown the presence of the markers of early differentiation in the derivates of three germ layers, characteristic for human ESC, which in corresponding microenvironments may allow MSC to be useful for reparation of tissue injures. The directed osteogenic and chondrogenic differentiation of line SC6-MSC has shown. However, no directed adipogenic differentiation of this line has been found. The obtained results with high probability may indicate what alteration of chromosomal and, accordingly, gene balance, in line SC6-MSC with karyotype 45, X0 resulted in decrease in differential potential, in expression CD90, associated in particular with the processes of differentiation and aging of cells.

[CHARACTERISTIC OF THE CELLULAR SPHEROIDS, DERIVED FROM MESENCHYMAL STEM CELL LINES FROM BONE MARROW AND MUSCLE OF LIMB OF EARLY HUMAN EMBRYO].

Cellular spheroids were derived from mesenchymal stem cell lines derived from 5-6-weeks embryo from different tissues of 5-6-week human embryo: bone marrow (FetMSC) and muscle of limb (M-FetMSC). Comparative analysis of the characteristics of these lines has been performed with 2D culturing in monolayer and 3D culturing in spheroids. The characteristics of cellular spheroids were obtained after 48 h after their formation from monolayer cultures on the 6th passage after decryopreservation. Spheroids in contrast to monolayer cultures are heterogeneous cell populations composed of fibroblast-like and epithelioid cells. Two-day spheroids are actively proliferating structure. Cell surface markers were analyzed using flow cytometry. Both in the monolayer cultures and cellular spheroids, this analysis has revealed the presence of expression of surface antigens CDD44, CD73, CD9O, CD105, HLA-ABC that are characteristic of human MSC, and the absence of expression if CD34 and HLA-DR. Nevertheless, the level of expression of CD90 and CD105 antigens was significantly lower in the spheroids as compared with corresponding monolayer cultures. Immunofluorescence and flow cytometry analysis of the expression of transcriptions factors and surface antigens characteristic of human embryonic stem cells showed the presence of expression of Sox-2 and SSEA-4 in 2D and 3D cultures. Lack of expression of Oct-4 in 2D cultures and its significant increase in 3D cultures has been found. Immunofluorescence analysis showed the presence of the markers of early differentiation in the derivates of three germ layers characteristic of human embryonic stem cells in the cellular spheroids of both lines, which coincides with 2D cultures of these lines. The directed osteogenic, chondrogenic and adipogenic differentiation of these lines has been shown. However, a number of differences has been found between monolayer cultures and spheroids. Adipogenic differentiation was more active in the cellular spheroids from cell line M-FetMSC a compared with corresponding monolayer cultures. Differences between the 2D and 3D cultures of both lines have been shown by the character of chondrogenic differentiation. The results obtained confirm the status of MSC for the cellular spheroids derived from monolayer cultured of cell lines FetMSC and M-FetMSC and apparently indicate a partial extension of their differentiation capacity as compared to monolayer cultured.

[Dynamics of the cell cycle in human endothelial cell culture infected with influenza virus].

Cell cycle in a culture of endothelial cells EAhy 926 infected with influenza virus was investigated. Cytometric analysis of culture, synchronized using contact inhibition, has shown that the exposure to the influenza virus in cells EAhy 926 lengthened S-phase of the cell cycle. This result has been tested and proven on culture EAhy 926 treated with nocodazole. Compared with lung carcinoma cells A549, in which influenza virus provokes the arrest of G0/G1 phase of the cycle, elongation of S-phase of cycle at a similar infection of endothelial culture EAhy 926 indicates that the influenza virus differently affects the dynamics of the cell cycle according to the origin of the infected culture.

[INDUCTION OF DECIDUAL DIFFERENTIATION OF ENDOMETRIAL MESENCHYMAL STEM CELLS].

In this study, we compared the ability of human mesenchymal stem cells derived from menstrual blood (eMSCs) and mesenchymal stem cells (MSCs) from other tissues to differentiate into decidual cells in vitro. It was demonstrated that during differentiation secretion of decidualization markers (prolactin and insulin-like growth factor binding protein-1) increases in eMSCs from adipose tissue (MSC-AD). Thus, the ability of eMSCs to differentiate into decidual cells is much higher than MSC-BM or MSC-AD. It makes eMSCs promising for application in cellular therapy of infertility associated with decidualzation insufficiency.

[CHARACTERISTIC OF ENDOMETRIAL MESENCHYMAL STEM CELLS IN CULTURE OBTAINED FROM PATIENT WITH ADENOMYOSIS].

Adenomyosis is form of endometriosis, common diseases of female reproductive system, which can lead to infertility in women. in this study we are obtained and characterized cell line endometrial mesenchymal stem cells from a patient with adenomyosis, and compare obtained cells with the cell line of healthy donor. Aim of this study was to assesses the extent of differences between cells from donor with adenomyosis and cells from healthy donor. Was established that compared lines had morphology like fibroblasts, were differentiated in adipocytes, were expressed mesenchymal markers and didn't expressed haematopoietic markers. Cytogenetic analysis of differentially stained metaphase chromosomes on G-banding (passage 6-7) showed that healthy donor's cells had predominantly normal karyotype. The cellular line from a patient with diagnosis of "adenomyosis" had a lot of cells with changes in karyotype's structure. These changes were related with aneuploidy of cellular population and the presence non-random chromosomal breaks, often in chromosomes 7 and 11. Analysis of this data allows the cells from adenomyosis characterized physiological stability in culture and karyotypic instability with non-random involvement certain chromosomal set. The cellular line obtained from donor with adenomyosis showed signs destabilization of he genome, typical for cell transformation. Division of adenomyosis cells to the 26th passage is stopped and these cells entered into a phase of replicative aging. Based on this, we can conclude that founded karyotype's hanges do not lead to transformation and immortalization of cells in vitro.

[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.

[Comparative characteristics of the stem cells isolated from subcutaneous and subepicardial adipose tissue].

BACKGROUND: Stem cells (SCs) considerably vary in morphological, immunophenotypic, proliferative, and differentiation characteristics depending on their tissue source. The comparative analysis of their biological properties is essential for the optimal choice of SCs for regenerative therapies. METHODS: Using immunocytochemistry, flow cytometry, histochemistry and real-time RT-PCR, we have investigated SCs obtained from human subepicardial (SEC-AT) and subcutaneous (SC-AT) adipose tissue and cultured under similar culture conditions without any differentiation-promoting factors. RESULTS: The cultures were similar in the high proportion of proliferating cell nuclear antigen (PCNA)-positive cells. In both cultures, immunophenotyping has revealed high expression of mesenchymal stem cell surface markers CD29, CD44, CD73, and CD105, low expression of CD31, CD34 and CD45, and wide variability in CD117, CD146 and CD309 expression. The only distinction in CD marker profile was significantly lower expression of CD90 in SCs from SEC-AT. Histochemical analysis has shown the lack of Oil Red O-positive cells in both cultures and about ten-fold higher number of alkaline phosphatase-positive cells among SCs from SC-AT. In the both cultures, immunocytochemistry has detected similar low expression of slow myosin heavy chain marker MAB1628 and smooth muscle actin marker α-hSMA. Gap junctional protein Connexin-43 expression was markedly higher in SCs from SC-AT, and epithelial cell marker Cytokeratin-19 expression was detected only in these cells. By RT-PCR, GATA4 mRNA was found to be highly expressed only in SCs from SEC-AT. CONCLUSIONS: Our results suggest that SC-AT, as compared with SEC-AT, is richer in epithelial cell and osteogenic progenitors. In turn, SEC-AT possesses cardiomyogenic SCs, and can be considered as an alternative to SC-AT as a source of SCs for cell cardiotherapy.

[The STAT5 signaling in the expression of alpha-subunit of interleukin-2 receptor in human blood lymphocytes].

The comparative study of the STAT3 and STAT5 activity (as assessed by tyrosine phosphorylation level) and the expression of a α-subunit of interleukin-2 receptor (as examined by cytophotometric evaluation of the number of CD25+ cells) during the phytohemagglutinin (PHA)-induced proliferation of human blood lymphocytes (HBL) have been made. It has been revealed that the level of STAT3 phosphorylation is high in both res ting and competent HBL and remains unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen in both resting and competent HBL. We observed phosphorylation of STAT5 no earlier than 5 h after PHA stimulation and the maximum phosphorylation was detected following 24 h. Exogenous IL-2 induced high level of STAT5 phosphorylation in the competent HBL as early as at 30 min and this level of STAT5 phosphorylation kept in the next 24-48 h. The correlation between alterations in tyrosine phosphorylation level of STAT5 and the expression of CD25 has been established. WHI-P131, an inhibitor of JAK3 kinase, prevents STAT5 activation, cell surface expression of CD25 and lymphocyte proliferation. It has been concluded that JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity αßγ(c)-receptor of IL-2 and optimal proliferation of HBL.

[Ability of the influenza viruses and their envelope proteins to stimulates endothelial cells apoptosis in vitro].

The ability of the modern epidemic strains of influenza virus type A (subtypes H5N1, H3N2, H1N1pdm) and their surface proteins, hemagglutinin (HA) and neuraminidase to cause the activation of the cellular protein caspase-3 and stimulate the emergence of phosphatidylserine on the membrane of human endothelial cell line EAhy926 has been studied. It was questioned how the viruses and their surface proteins that were studied cause the activation of caspase-3 after 0.5 h of exposure that recorded immunogistotsitohimicheski. The test viruses and neuraminidase (concentration 10 µg/ml) led to the appearance of phosphatidylserine on the cell membrane in a time interval of 2-8 h from the beginning of the treatment, which was recorded by flow cytometry. The death of endothelial cells when exposed to the HA (in a concentration of 50 µg/ml) and in the same time frame was not accompanied by the appearance of phosphatidylserine. The specific feature of apoptotic cell death during the reproduction of the virus are described, as well as the effects of viral proteins.

[Mesenchymal stem cells of human endometrium do not undergo spontaneous transformation during long-term cultivation].

Mesenchymal stem cells isolated from human endometrium (eMSC) are perspective source of stem cells for regenerative medicine. Large amount of these cells accumulated by in vitro cultivation is usually required for transplantation into patients. We established several cell eMSC lines and cultivated them during long period of time to examine the possibility of their spontaneous transformation. All cell lines demonstrate limited lifespan, undergo replicative senescence and die. Karyotypic analysis on different passages reveals that most cells display karyotypic stability. Thus, extended in vitro cultivation of eMSCs does not lead to spontaneous transformation that makes therapeutic application of these cells safety for patients. During long-term cultivation eMSCs sustain the expression of surface markers.

[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.

[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.

[The cell cycle regulator p130 and beta-catenin form a complex in mesenchymal stem cells].

Suppressor complex p130/E2f4 inhibits transcription of multiple genes proteins regulating cell cycle progression and induces cell cycle arrest at G0/G1 required for induction of cell differentiation in cells of many tissues in vivo and various cell lineages in vitro. We found here that, in mesenchymal stem cells, (MSC) activation of the Wnt/beta-catenin signal pathway induced by MSC coculture with the A-549 cell line or by growth in the medium containing Li+ ions, which resulted in the accumulation of active forms of the p130, E2f4 and beta-catenin, was not coupled with inhibition of cell cycle progression. Cell cycle synchronization of the MSC induced by thymidine and nocodazol was not resulted in change of the levels and phosphorylation pattern of the p130 in contrast to mouse hepatocytes and T98G cells which showed accumulation of the p130 form p1 and p2 in quiescence and form p3 under active proliferative. Antibody to p130 precipitated from extracts of MSC activated by Li+ ions beta the p130 form 2 and hyperphosphorilated beta-catenin. The results obtained suggest that Gsk3beta, p130 and beta-catenin form in MSC a complex the functional role of which may be associated with activation of differentiation not coupled to cell cycle arrest.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. I. The role of JAK and Src tyrosine kinases as revealed by inhibitory analysis].

The long-lasting expression of an alpha-subunit of interleukin-3 receptor (IL-2Ralpha) was found to accompany the PHA-induced proliferation of human blood lymphocytes (HBL), so that to the end of the second day of mitogenic stimulation only, the large blasts may express the high affinity alphabetagamma(c) receptor for IL-2. With the selective pharmacological drugs to JAK (WHI-P131) and Src (PP2) it is shown that the non-receptor tyrosine kinases are involved in the surface CD25 expression. It is revealed that the PP-2-inhibitable expression of CD25 is timely associated with the initial stage of T cell activation, whereas WHI-P131-inhibitable expression was present during the whole G0/G1/S transition. These data indicate that at the early, antigen-dependent stage the expression of IL-2Ralpha is induced via Src-dependent signaling pathway, and prolonged increase in IL-2Ralpha expression is regulated by IL-2/IL-2 receptor interaction via JAK-dependent signaling pathway.

[The surface expression of CD25 at different stages of proliferative response in human lymphocytes. II. The role of interleukin-2].

The expression of alpha-subunit of interleukin-2 receptor (IL-2Ralpha) was assessed by quantifying activation-induced upregulation of CD25 in human blood lymphocytes (HBL) stimulated by interleukin-2 (IL-2). It was established that exogenous IL-2 induced no surface expression of CD25 neither proliferation at 48 h of IL-2 action. In component HBL, pretreated by sub-mitogenic doses of phytohemagglutinin (PHA), 5-15 % of cell population was revealed to represent the CD2t+ cells, and in the competent cells only, exogenous IL-2 induced the surface expression of CD25 as well as the growth and the proliferative response, which was comparable with those to mitogenic doses of PHA. The JAK3 inhibitor WHI-P131 eliminated IL-2-dependent CD25 expression without influencing the CD25 expression in competent cells. Unlike, PP2 was found to inhibit the IL-2-dependent CD25 expression in a lesser extent than WHI-P131, however this drug was effectively inhibited CD25 expression in PHA-pretreated, competent HBL. These data suggest that Src-dependent signaling participate in the early IL-2Ralpha expression that precedes the IL-2-dependent cell cycle progression of activated HBL. It is concluded that in normal T cells, the IL-2Ralpha expression in firstly induced by antigen (mitogen) and thereafter it is held IL-2 through JAK-dependent signaling pathway.

[Effect of formaldehyde in low concentrations on the proliferation and organization of the cytoskeleton of cultured cells].

3-4% (1.07-1.42 M) formaldehyde is one of the most popular and well-known organs, tissues and cells fixer. In this manuscript we have shown that formaldehyde in concentrations of up to 60 microM (0.0002%) does not have any negative effect on the viability of cell lines A431, HEK293 and primary rat fibroblasts, but it is also increases the proliferative activity of A431. The influence on A431 cells might be explained by the activation of epidermal growth factor receptors as a result of their interaction with formaldehyde.

[Multipotent mesenchymal stem cells of desquamated endometrium: isolation, characterization and use as feeder layer for maintenance of human embryonic stem cell lines].

In this study, we characterize new multipotent human mesenchymal stem cell (MSC) lines derived from desquamated (shedding) endometrium in menstrual blood. The isolated endometrial MSC (eMSC) is an adhesive to plastic heterogeneous population composed mainly of endometrial glandular and stromal cells. The established cell lines meet the criteria of the International Society for Cellular Therapy for defining multipotent human MSC of any origin. The eMSCs have positive expression of CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II). Multipotency of the established eMSC is confirmed by their ability to differentiate into other mesodermal cell types such as osteocytes and adipocytes. Besides, the isolated eMSC lines partially (over 50%) express the pluripotency marker SSEA-4, but do not express Oct-4. Immunofluorescent analysis of the derived cells revealed the expression of the neural precursor markers nestin and beta-III-tubulin. This suggests a neural predisposition of the established eMSC. These cells are characterized by high rate of cell proliferation (doubling time 22-23 h) and high cloning efficiency (about 60%). In vitro the eMSCs undergo more than 45 population doublings revealing normal karyotype without karyotipic abnormalilies. We demonstrate, that the mititotically inactivated eMSCs are perfect feeder cells for human embryonic stem cell lines (hESC) C612 and C910. The eMSC being a feeder culture maintain the pluripotent status of the hESC, which is revealed by the expression of Oct-4, alkaline phosphatase and SSEA-4. When co-culturing, hESC retain their morphology, proliferative rate for more than 40 passages and capability for spontaneous differentiation into embryoid bodies comprising the three embryonic germ layers. Thus, an easy and non-invasive extraction of the eMSC in menstrual blood, their multipotency and high proliferative activity in vitro without karyotypic abnormalities demonstrate the potential of use of these stem cells in regenerative medicine. Using the derived eMSCs as the feeder culture eliminates the risks associated with animal cells while transferring hESC to clinical setting.

[Structure of neuromuscular junctions and differentiation of striated muscle fibers of mdx mice after bone marrow stem cells therapy].

Mdx mice are a model of Duchenne muscular dystrophy caused by deficiency of dystrophin. Muscles of mdx mice are characterized by high levels of striated muscle fibers death and, accordingly, by a high level of its regeneration. Moreover, the structure of neuromuscular junctions in mdx mice is altered. Changes in the structure of mdx mice neuromuscular junctions against a background of increasing differentiation of striated muscle fibers after C57BL/6 Lin (-) bone marrow stem cells transplantation were investigated. The muscles were studied in 4, 8, 16 and 24 weeks after transplantation. We observed that the level of striated muscle fibers loss was decreased from the 4th week after transplantation of bone marrow stem cells. Accumulation of muscle fibers without centrally located nuclei began from the 8th week, and dystrophin synthesis was increased at the 16th and 24th weeks after bone marrow stem cells transplantation. Longitudinal sections of quadriceps muscles of mdx mice showed decrease in the number of acetylcholine receptors clusters in neuromuscular junctions and a simultaneous increase in acetylcholine receptor clusters area during the 4th week after transplantation. In 16 weeks after bone marrow stem cells transplantation, total neuromuscular junction area was increased due to increase in the area of acetylcholine receptors clusters and to increase in their number as well. Thus, single intramuscular transplantation of C57BL/6 Lin (-) bone marrow stem cells induces an increase in differentiation of mdx mice striated muscle fibers and improves the structure of neuromuscular junctions.

[The expression of CD25 in phytohemagglutinin- or interleukin-2-stimulated human peripheral blood lymphocytes].

The timely expression of a high affinity receptor for interleukin-2 (IL-2R) in human peripheral blood lymphocytes stimulated by various mitogens was examined by cytophotometric evaluation of the number of CD25 bearing cells (CD25+). In resting lymphocyte culture both phytohemagglutinin (PHA, 10 (microg/ml) and 12,13-phorbol dibutirate (PDBu, 10-8 M) and ionomycin (IM, 5 x 10(-7) M) induce the long-lasting increase (during 48 h) in the number of CD25+ cells. Only in competent (not in resting) lymphocytes, pretreated by submitogenic doses of PHA (1 microg/ml), interleukin-2 (IL-2) is capable to induce the time-dependent CD25 expression. When comparing the time course of CD25+ expression and the blasttransformation it was found that the CD25 markers were revealed on small stimulated lymphocytes and all the large blasts were the CD+ cells with high-affinity alphabetagamma(c) receptor for IL-2. In conclusion, the expression of alpha-subunit of IL-2R takes place during the IL-2-dependent stage of T cell proliferation and may be directly induced by IL-2 via IL-2R.

[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.