GC-Rich Extracellular DNA Induces Oxidative Stress, Double-Strand DNA Breaks, and DNA Damage Response in Human Adipose-Derived Mesenchymal Stem Cells

Figure 6

GC-DNA induces adipogenic differentiation of haMSCs. HaMSCs were grown to subconfluency (~80%) and then DNA samples were added. Cells were cultivated in the presence of DNA samples (50 ng/mL) at 37°C for 14 days in AmnioMax Basal Medium with AmnioMax Supplement C100 (Gibco). In a week the culture medium with DNA samples was refreshed. (a) Changes in the morphology of the cultured cells (100x). Cells were fixed with isopropanol and stained with crystal violet. Control (−) DNA samples were not added to the medium; control (+) cells were cultured in the medium reliably inducing adipogenesis (StemCell Technologies Inc.). (b) Adipogenic differentiation of haMSCs was identified by fixing the cells with 4% PFA and staining with 0.3% Oil Red O solution and with CytoGreen (20x). (c) Relative levels of RNA for PPARG2 and LPL. The cells were fixed after 7 days of incubation. (d) Detection of FABP4 protein level in haMSCs exposed to DNA samples. Cells were fixed with 3% PFA, treated with 0.1% Triton X-100 and stained with anti-FABP4 (FITC) antibodies. (A) FACS analysis showing the stained cellular fractions (SSC-FL1 (FABP4) diagram). Gate R encircles the fraction of haMSCs that express large amounts of protein FABP4; (B) the distribution of fluorescence intensities of the cells stained with anti-FABP4 (FITC) antibodies; (C) the proportion of cells with large amounts of protein FABP4 (gate R). Horizontal bars reflect relative expression levels in the control cells. Data points were averaged and represented as mean ± SD for three biological replicates. Asterisk   depicts the differences between exposed cells and control cells that were statistically significant by the Mann-Whitney U test ().

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