DERIVATION AND CHARACTERISTICS OF THE NEW SUBLINE OF HUMAN EMBRYONIC STEM CELLS SC6-FF IN ALLOGENIC FEEDER-FREE CULTURE SYSTEM.

A new human embryonic stem cell subline SC6-FF has been derived from SC6 cells in allogenic feeder-free culture system. Extracellular matrix proteins and conditioned medium from mesenchymal stem cell line SC6-MSC were the key components of the feeder-free culture system that therefore was allogenic for SC6-FF cells. SC6-FF subline has underwent more than 100 cell population doublings and retained normal diploid human karyotype: 46, XX. The average doubling time of the cell population was 23.7 ± 0.8 h that does not differ from that for the parent SC6 line. The presence of undifferentiated hESCs markers, alkaline phosphatase activity, Oct-4, SSEA-4 and TRA-1-60, has been verified by histochemical and immunofluorescence analysis. Non-directional differentiation of SC6-FF subline has led to development of cells that differ in size and morphology from the cells in the parent population. These cells demonstrate the ability of differentiation in the derivates of three germ layers by expressing the characteristic markers of the ectoderm (alpha-fetoprotein), mesoderm (a-actinin) and endoderm (a-fetoprotein) cells. We can conclude that the obtained characteristics of the new feeder-free SC6-FF sub-line correspond to the status of the human embryonic stem 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.

[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.

Proliferation and survival of rat C6 glioma culture in the presence of implants coated with modified carbon-based films.

The survival of rat C6 glioma decreased in the presence of implants from VT-16 titanium alloy. Diamond-like carbon coating of VT-16 alloy slightly increased cell death on day 5 of the experiment (39.9+/-2.1%). The percentage of dead C6 glioma cells inside titanium rings with diamond-like carbon coating, incorporating up to 3.5 atom.% Ag nanoparticles, was 53.7+/-4.3% on day 5 of culturing, while after doping to 6.7 atom.% Ag cell death reached 66.7+/-3.2% (p<0.05). The maximum toxic effect towards C6 glioma was detected in the specimens coated with diamond-like film with silver nanoparticles.