TGF-β1 induces senescence of bone marrow mesenchymal stem cells via increase of mitochondrial ROS production

Abstract

Background: Bone marrow derived mesenchymal stem cells (bmMSCs) are multipotent cells that can differentiate into diverse cell types, including cardiomyocytes. BmMSC-based transplantation is capable of repairing acute and chronic myocardial infarction. Prior to the transplantation, MSCs are usually induced in vitro by biological reagents and chemicals for directional differentiation. Transforming growth factor beta (TGF-β) is one of the most commonly used biological reagents for induction of cardiomyocyte differentiation of bmMSCs. Previous studies have shown that TGF-β induces senescence in several cell types. However, whether TGF-β affects senescence of bmMSCs has not been elucidated. The goal of this study was to investigate the effect of TGF-β1 on senescence of bmMSCs and the underlying mechanisms.

Results: We found that TGF-β1 increased activity of senescence-associated-galactosidase (SA-Gal) and production of mitochondrial reactive oxygen species (mtROS) in bmMSCs in a dose-dependent manner. TGF-β1 also significantly decreased expression of superoxide dismutase 2 (SOD2) and Id1, and increased expression of 4-Hydroxynonenal (4-HNE) subunits and p16 in bmMSCs in a dose-dependent manner. Pre-treatment with mtROS inhibitor acetyl-L-carnitine (ALCAR, 0.1 mM) significantly inhibited TGF-β1-induced mtROS production and SA-Gal activity.

Conclusion: TGF-β1 can induce senescence of bmMSCs, which at least partially depends on mtROS production.

Figure 1

Identification of bone marrow mesenchymal stem cells (bmMSCs). Immunofluorescence assay shows that bmMSCs positively express MSC markers CD44 and CD90. Immunochemistry staining shows that alkaline phosphatase (ALP) is positively expressed in bmMSCs following 3 weeks of osteogenic differntiation culture. Oil Red O staining displays that lipid droplets are accumulated in a part of bmMSCs following 3 weeks of adipogenic differentiation culture.

Figure 2

Dose–response of β-galactosidase activity in bone marrow mesenchymal stem cells (bmMSCs) after exposure to 0, 1, 5 and 10 ng/mL recombinant mouse transforming growth factor β1 (TGF-β1) for 24 hours. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. control.

Figure 3

Time-response of β-galactosidase activity in bone marrow mesenchymal stem cells (bmMSCs) after exposure to 5 ng/mL recombinant mouse transforming growth factor β1 (TGF-β1) for 0–24 hours. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. control.

Figure 4

Western-blot assay shows expression of aging marker proteins 4-Hydroxynonenal (4-HNE), p16 and Id1 in bmMSCs after exposure to 0, 1, 5 and 10 ng/mL TGF-β1 for 24 hours. A. 4-HNE expression; B. P16 expression; C. Id1 expression. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. control.

Figure 5

Mitochondrial reactive oxygen species (mtROS) production in bone marrow mesenchymal stem cells (bmMSCs) after exposure to 0, 1, 5 and 10 ng/mL TGF-β1 for 24 hours. A. MitoSOX™ Red Indicator staining shows mtROS in bmMSCs after exposure to 0, 1, 5 and 10 ng/mL TGF-β1 for 24 hours. B. Quantification of fluorescence density of mtROS. C. Western blot assay shows expression of superoxide dismutase 2 (SOD2) in bmMSCs after exposure to 0, 1, 5 and 10 ng/mL TGF-β1 for 24 hours. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. control.

Figure 6

Mitochodrial ROS (mtROS) specific inhibitor acetyl-L-carnitine (ALCAR) inhibits TGF-β1-induced mtROS generation and bmMSC senescence. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. treatments with 5 ng/mL TGF-β1.

Figure 7

Expression of aging markers 4-Hydroxynonenal (4-HNE), p16 and Id1 in bmMSCs as the cells were exposed to acetyl-L-carnitine (ALCAR, 0.1 mM) and TGF-β1 (5 ng/mL) simultaneously. A. 4-HNE expression; B. P16 expression; C. Id1 expression. Bar graphs represent mean ± SD (4 independent experiments/group). *P < 0.05 vs. treatments with 5 ng/mL TGF-β1.