Macrophage migration inhibitory factor rejuvenates aged human mesenchymal stem cells and improves myocardial repair

Figure 1. Aged MSCs display increased cellular senescence. (A) Representative images of young and aged MSCs under light microscopy, and quantitative analysis of the cell size of young and aged MSCs. (B) Representative images of SA-β-gal staining, and quantitative analysis of SA-β-gal-positive cells among young and aged MSCs. (C) Western blotting and quantitative analysis of p53 and p21 protein expression in young and aged MSCs. (D) Representative images of Ki-67 staining and quantitative analysis of Ki-67-positive cells among young and aged MSCs. (E) Representative images of the wound healing assay and quantitative analysis of the wound recovery rate in young and aged MSCs. Scale bar=100 μm. Data are expressed as the mean±SEM. n=3. **p<0.01; ***p<0.001.

Figure 2. MIF inhibited the cellular senescence of MSCs. (A) Western blotting and quantitative analysis of MIF protein expression in young and aged MSCs. (B) Western blotting and quantitative analysis of MIF, p53 and p21 protein expression in young MSCs treated with control siRNA or MIF-siRNA. (C) Representative images of SA-β-gal staining and quantitative analysis of SA-β-gal-positive cells in young MSCs treated with control siRNA or MIF-siRNA. (D) Western blotting and quantitative analysis of MIF, p53 and p21 protein expression in aged and MIF-aged MSCs. (E) Representative images of SA-β-gal staining and quantitative analysis of SA-β-gal-positive cells in aged and MIF-aged MSCs. (F) Cell growth curves showed that overexpression of MIF in aged MSCs increased their growth rate. Scale bar=100 μm. Data are expressed as the mean±SEM. n=3. **p<0.01; ***p<0.001.

Figure 3. MIF rejuvenated aged MSCs by promoting autophagy. (A) Western blotting and quantitative analysis of LC3I/II, Beclin1, p62, p53 and p21 protein expression in aged MSCs and MIF-aged MSCs with or without 3-MA treatment. (B) Representative images of TUNEL staining and quantitative analysis of the apoptosis of aged MSCs and MIF-aged MSCs with or without 3-MA treatment under an SD/H challenge. Scale bar=100 μm. Data are expressed as the mean±SEM. n=3. **p<0.01; ***p<0.001.

Figure 4. Transplantation of MIF-aged MSCs improved cardiac function in a rat model of MI. (A) Representative images of M-mode echocardiography captured 28 days after MI in rats from the different groups. (B) Heart function, including the LVEF and LVFS, was evaluated at 0, 7 and 28 days in control mice and MI mice with or without MSC transplantation. (C) Representative images of Masson's Trichrome staining and quantitative analysis of heart fibrosis in the different experimental groups. Scale bar=2 mm. Data are expressed as the mean±SEM. n=6-7. **p<0.01; ***p<0.001.

Figure 5. MIF overexpression enhanced cell survival and angiogenesis in the rat heart after MI. (A) Representative images of HNA staining and quantitative analysis of MSC survival in the heart tissue of rats in the different groups at 28 days. Scale bar=50 μm. (B) Representative images of α-SMA staining in the heart tissue of rats from the different groups at 28 days. Scale bar=100 μm. (C) Quantitative analysis of the α-SMA density in the heart tissue of rats from the different groups at 28 days. (D) Representative images of CD31 staining in the heart tissue of rats from the different groups at 28 days. Scale bar=100 μm. (E) Quantitative analysis of the CD31 density in the heart tissue of rats from the different groups at 28 days. Data are expressed as the mean±SEM. n=6-7. **p<0.01; ***p<0.001.