Simulated Microgravity Condition Alters the Gene Expression of some ECM and Adhesion Molecules in Adipose Derived Stem Cells

Abstract

Adipose- derived stem cells (ADSCs) are widely used for tissue engineering and regenerative medicine. The beneficial effects of ADSCs on wound healing have already been reported. Remodeling of extracellular matrix (ECM) is the most important physiological event during wound healing. ECM is sensitive to mechanical stresses and the expression of its components can be therefore influenced. The aim of this study was to investigate the effect of simulated microgravity on gene expression of some ECM and adhesion molecules in human ADSCs. After isolation and characterization of ADSCs, cells were exposed to simulated microgravity for 1, 3 and 7 days. Real-time PCR, fluorescence immunocytochemistry, and MTT assay were performed to evaluate the alterations of integrin subunit beta 1 (ITGB1), collagen type 3 (ColIII), matrix metalloproteinase-1 (MMP1), CD44, fibrillin (FBN1), vimentin (VIM) genes, and ColIII protein levels as well as cells viability. Microgravity simulation increased the expression of ITGB1, ColIII, MMP1, and CD44 and declined the expression of FBN1 and VIM genes. ColIII protein levels also increased. There were no significant changes in the viability of cells cultured in microgravity. Since the high expression of ECM components is known as one of the fibroblast markers, our data suggest that pretreatment of ADSCs by simulated microgravity may increase their differentiation capacity towards fibroblastic cells. Microgravity had not adversely affected the viability of ADSCs, and it is likely to be used alone or in combination with biochemical inducers for cell manipulation.

Keywords: Adipose- derived stem cells; adhesion molecules; extracellular matrix; simulated microgravity.

Fig. 1

Isolated ADSCs with the enzymatic method from lipoaspirate samples. (A). Following a 48- hour SVF culture, fibroblast-like spindle shape cells can be clearly observed (100x).The arrows indicate the remains of oil spots from digested fat. (B). After changing medium, ADSCs at 40-50% confluence were observed. Magnification 100 x

Fig. 2

Characterization of ADSCs. Figures A to F show the ability of human ADSCs to differentiate into adipocyte and osteoblast lineages (100x). (A and D) ADSCs cultured in control media; (B and C) ADSCs cultured in the presence of adipogenic inducer for 9 days and stained with Oil Red O dye. Arrows show the accumulation of lipid vacuoles indicating differentiation to adipogenic cell lineage; (E and F) ADSCs cultured in the presence of osteogenic inducer for 15 days and stained with Alizarin Red S dye. Production of orange-red calcium deposits demonstrated the successful differentiation of ADSCs to osteogenic cell lineage. (G) Immunophenotypic characterization of human MSCs were carried out using flow cytometry. Third-passage of isolated ADSCs was positive for MSCs markers including CD90, CD73 and CD105 and negative for hematopoietic markers including CD34 and CD45 (for all the donors)

Fig. 3

Cell viability of ADSCs cultured in simulated microgravity (SMG) and static culture after 7 days. There were no statistically significant changes in viability between two culture conditions (n=3)

Fig. 4

Relative gene expression analysis of some ECM and adhesion molecules under simulated microgravity condition for the 1, 2 and 7 days in comparison to the control group in normal gravity (n=3). Gene's expressions were normalized to GAPDH in the same samples. Values are mean ± staandard deviation; *indicates P<0.05

Fig. 5

Fluorescence immunocytochemistry analysis of ColIII. (A and B) Anti-ColIII staining in cells cultured in normal or 1g condition. (C and D) Anti- ColIII staining in cells cultured in simulated microgravity condition for 7 days. (A and C) Anti- ColIII staining without Hoechst treatment. (B and D) Anti-ColIII staining plus Hoechst treatment. The circle shows cell aggregated production. ImageJ was used to quantify the COLIII-positive area in six randomly chosen fields for each experiment (n =3). P<0.05 was considered statistically significant. Magnification 100x.