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. 2017:2017:5841716.
doi: 10.1155/2017/5841716. Epub 2017 Jun 21.

SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation

Ryan A Denu  1
Affiliations

SIRT3 Enhances Mesenchymal Stem Cell Longevity and Differentiation

Ryan A Denu. Oxid Med Cell Longev. 2017.

Abstract

Mesenchymal stem cells (MSCs) are multipotent cells that are currently being investigated in a wide variety of clinical trials for their anti-inflammatory and immunomodulatory properties as well as their osteogenic and chondrogenic capabilities. However, there are considerable interdonor variability and heterogeneity of MSC populations, making it challenging to compare different products. Furthermore, proliferation, differentiation, and immunomodulation of MSCs decrease with aging and ex vivo expansion. The sirtuins have emerged as a class of protein deacylases involved in aging, oxidative stress, and metabolism. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase involved in reducing oxidative stress while preserving oxidative metabolism, and its levels have been shown to decrease with age. This study investigated the role of SIRT3 in MSC differentiation and aging. As MSCs were expanded ex vivo, SIRT3 levels decreased. In addition, SIRT3 depletion reduced MSC differentiation into adipocytes and osteoblasts. Furthermore, overexpression of SIRT3 in later-passage MSCs reduced aging-related senescence, reduced oxidative stress, and enhanced their ability to differentiate. These data suggest that overexpressing SIRT3 might represent a strategy to increase the quality and quantity of MSCs utilized for clinical applications.

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Figures

Figure 1
Figure 1
Immunophenotype of MSCs. (a) MSCs were isolated from bone marrow aspirates of 3 healthy donors. Representative flow cytometry histograms are shown. The black line indicates cells stained with the indicated antibody, while the gray histogram indicates cells stained with the isotype control. Passage 3 cells were used. (b) A bar graph demonstrating the average percent of positive cells for 3 MSC strains ± SEM.
Figure 2
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 bar = 100 μm. (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 bar = 100 μm. (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 (CT) 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. ∗P < 0.05 from one-way ANOVA tests.
Figure 3
Figure 3
SIRT3 increases with MSC differentiation. (a) Western blotting demonstrating SIRT3 expression as 3 strains of passage 3 MSCs are differentiated into adipocytes and osteoblasts using appropriate induction media for 21 days. (b) Quantification of protein expression in western blots from panel (a). Expression was quantified using ImageJ and normalized to the expression of untreated MSCs for each of the 3 strains. (c) Quantitative RT-PCR demonstrating SIRT3 mRNA expression in 3 strains of MSCs after 21 days of exposure to appropriate differentiation media. Cycle threshold (CT) values were normalized to the combination of 3 housekeeping genes (RRN18S, GAPDH, and ACTB). Bars represent means ± SEM of 2−ΔCT values. ∗P < 0.05 from two-sided t-tests comparing adipocytes and osteoblasts to undifferentiated MSCs.
Figure 4
Figure 4
SIRT3 is required for MSC differentiation. (a) Passage 3 MSCs were transduced with retrovirus expressing shRNA against SIRT3 or scrambled control. Two different SIRT3 shRNA sequences were used. Western blotting was used to demonstrate efficient depletion of SIRT3. (b) To assess senescence, β-galactosidase activity assay was performed. Bars represent means ± SEM for the 3 MSC strains. ∗P < 0.05 from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to both untreated and shScrambled conditions. (c) To assess ROS, MSCs were stained with DHE, which was detected by flow cytometry. Bars represent average DHE values (median channel fluorescence from FL3) ± SEM from the 3 MSC strains. “Untreated” refers to wild-type MSCs that were not virally transduced. ∗P < 0.05 from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to both untreated and shScrambled conditions. (d) MSCs were differentiated for 21 days with appropriate adipogenic or osteogenic induction media. Adipocytes were stained with oil red O, and osteoblasts were stained with alizarin red S. Representative images are shown. Wild-type MSCs not treated with differentiation media but still stained with either oil red O or alizarin red S served as controls. Scale bar = 100 μm. (e) Isopropanol was used to elute the oil red O and alizarin red S, and absorbance was read at 500 nm or 520 nm, respectively. Bars represent means ± SEM for 3 MSC strains. ∗P < 0.05 from two-sided t-tests comparing shSIRT3-1 and shSIRT3-2 to shScrambled.
Figure 5
Figure 5
SIRT3 overexpression restores differentiation and reduces oxidative stress in aged MSCs. (a) SIRT3 was overexpressed in later-passage (passage 7) MSCs compared to untreated later-passage MSCs and early-passage (passage 1) MSCs and subsequently assessed by western blotting. VEC: empty vector control. Alpha tubulin served as a loading control. (b) Differentiation was assessed 21 days after the treatment with appropriate induction media. Adipocytes were stained with oil red O, and osteoblasts were stained with alizarin red S. Scale bar = 100 μm. (c) Oil red O and alizarin red S were eluted with isopropanol; then, absorbance was read at 500 nm or 520 nm, respectively. (d) Senescence was assessed by quantifying the percentage of cells with β-galactosidase activity. Bars represent means ± SEM for the 3 MSC strains for 3 technical replicates of 1 MSC strain. “Untreated” cells were not transfected. (e) MSCs were stained with DHE, which was detected by flow cytometry. Bars represent average DHE values (median channel fluorescence from FL3) ± SEM from the 3 MSC strains. Cells treated with NAC, an ROS scavenger, served as a negative control. ∗P < 0.05 from two-sided t-tests.
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