CHARACTERISTIC OF THE CELLULAR SPHEROIDS, DERIVED FROM MESENCHYMAL STEM CELL LINES FROM BONE MARROW
AND MUSCLE OF LIMB OF EARLY HUMAN EMBRYO
T.A. Krylova, A.S. Musorina, V.V. Zenin, G.G. Poljanskaya 1
Institute of Cytology RAS, St. Petersburg, 194064;
1 e-mail: poljansk@mail.cytspb.rssi.ru
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.
Key words:
human mesenchymal stem cell lines, cellular spheroids immunofluorescence analysis, surface
cell markers, transcription factors, differentiation
| Back
| Contents
| Main |
