[ULTRASTRUCTURAL CHANGES OF THE STEM CELLS IN THE CYCLE MONOLAYER--SPHERES--MONOLAYER].

Sphere formation can be used to prepare stem cells (SCs) prior to transplantation. Here SCs isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer-spheres-monolayer cycle by transmission electron microscopy. The results obtained with both adherent-induced and hanging-drop induced spheres were similar. At first 2-3 passages (stage 1), isolated SCs displayed embryonal cell-like ultrastructure. With increasing passage times (stage 2), SCs became bigger and more electron-dark with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), prominent Golgi apparatus and numerous vacuoles. After 2 h from the initiation of the formation of spheres (stage 3), SCs gathered into clusters and formed desmosome-like intercellular contacts. Their nucleus possessed a large loose fibrillo-granular nucleoli, the cytoplasm was densely packed with disintegrated cisternae of RER, Golgi apparatus was not detected. After 24 h from the initiation of spheres (stage 4), SCs in well-formed spheres exhibited large dense nucleoli and poorly developed Golgi apparatus and RER. One day after sphere dissociating (stage 5), SCs were embryonal cell-like and morphologically similar to the cells of the first stage except for the presence of a large nucleolus and numerous Golgi complexes. After 48 h from sphere dissociating (stage 6), SCs became electron-dark and resembled the SCs of the second stage by the presence of irregularly shaped nuclei and the cetoplasm filled with RER. We interpreted the results as senescence of the SCs with the number of passages after isolation from tissue and a day after dissociation of the spheres and as rejuvenation of the SCs just after sphere dissociation. Further research is needed to reveal the genetic, biochemical and physiological parameters of the SCs on established morphologically distinct stages in order to provide higher-quality cellular material for disease cell therapy.

[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.

[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.

[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.

[Effect of type I collagen and fibronectin on cell morphology of human MSCs in vitro].

Limited knowledge about behaviour of stem cells in culture seems to be one of the reasons for problems in their successful introduction to applied medicine. To address this issue we have studied in vitro interaction of human mesenchymal stromal cells (MSCs) with various substrates (plastic, type I collagen, fibronectin, and mixtures of these proteins at various ratios) during the 16-18 h after cell plating. Several cell morphology features such as Area, Perimeter, spreading coefficient, polarization coefficient were determined. It has been shown that MSCs respond specifically to the substrate and can be classified into several groups according to the parameters studied. Collagen preferably fibronectin have opposite effects on polarization and spreading of the cells. Collagen preferably enhances polarization of the cells, whereas fibronectin stimulates proportional spreading of cells. Effect of collagen-fibronectin mixture on the cells cannot be considered as a simple additive effect. We assume that variation in the ratio of these proteins in the extracellular matrix might be one of the possible ways to influence the morphology of stem cells when they are induced to differentiate.

[Two spreading states of mesenchymal cells of the human embryo in vitro].

Spreading mesenchymal cells of human embryo on plastic and type I collagens (from rat, sheep and bull) was studied. Spreading of the cells on collagens was stronger than that in the control but no differences between the different collagens were revealed. The cell perimeter, the spreading coefficient and the cell projection area on the substrate were used as morphometric parametres. The spreading of cells was monitored for 0.5-2 h after plating. During the spreading both on plastic and on collagen, the groups of small cells were revealed as separate subpopulations. As a whole, such cells comprehend 9 % of the cell population in the control and 2% in experiment. We assume that this cell type is associated with a special independent functional state of the cells that precedes cell spreading.

[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.

[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.

[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.

[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.

[Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles]. / Monoklonal'nye antitela k myshechnoi izoforme al'fa-aktinina--marker dlia izucheniia differentsirovki skeletnoi i serdechnoi myshts.

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.

[T- and B-immunity system indices in candidiasis]. / Nekotorye pokazateli T- i B-sistem immuniteta u bol'nykh kandidozom.

The examination of 73 patients with superficial and visceral candidiasis and Candida-carriers yielded no data to the effect that the increased activity of opportunistic fungi of the genus Candida in the body was preceded by organic lesions in the T- and B-systems of lymphocytes. Only in 16 patients who had visceral candidiasis for 8-12 years with combined lesions of several organs the decreased capacity of lymphocytes for reacting with phytohemagglutinin was observed, which was probably a manifestation of the secondary immune insufficiency, developing in the presence of prolonged infection.