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, CD90, 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.