SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation

Figure 2

Differentiation capability and SIRT3 expression decrease with passaging. (a) To assess senescence, cells were assayed for β-galactosidase activity. Representative brightfield images of β-galactosidase staining in early- (passage 3) and late- (passage 9) passage MSCs. Scale . (b) The percentage of cells stained in blue (indicating they are senescent) was assessed in 3 technical replicates from one MSC strain. 24-hour treatment of passage 5 MSCs with 4 μM doxorubicin was used as a positive control for senescence. (c) Representative images of early- and late-passage MSCs after 21 days of treatment with appropriate differentiation media. Adipocytes were stained with oil red O, and osteoblasts were stained with alizarin red S. Scale . (d) Oil red O and alizarin red S were eluted with isopropanol; then, absorbance was read at 500 nm or 520 nm, respectively. Bars represent means ± SEM in 3 technical replicates from one MSC strain. (e) SIRT3 expression was assessed in 3 different MSC strains after passaging for 11 passages. Alpha tubulin was used as a loading control. (f) Protein level from western blots in panel (e) was quantified using ImageJ and normalized to the SIRT3 level in passage 3 cells in each strain. Bars represent means ± SEM for the 3 MSC strains. (g) SIRT3 mRNA was measured by qRT-PCR. Cycle threshold (C_T) values were normalized to the combination of 3 housekeeping genes (RRN18S, GAPDH, and ACTB). Relative mRNA values were calculated using 2^−ΔCT, which were then normalized to passage 3 (P3) values. Bars represent means ± SEM for 3 MSC strains. (h) ROS were assessed by staining with DHE and analyzing by flow cytometry. N-Acetyl cysteine (NAC) scavenges ROS and was used as a control. Bars represent average DHE values (median channel fluorescence from FL3) ± SD from 3 technical replicates from one MSC strain. from one-way ANOVA tests.

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