Figure 4

Effect of macrophage migration inhibitory factor on proliferation, paracrine signaling and survival of mesenchymal stem cells. (A) Proliferation growth curves of mesenchymal stem cells (MSCs), determined by the Cell Counting Kit-8 (HaiGene Technology, Harbin, China) assay, in young MSCs, aged MSCs and aged MSCs treated with macrophage migration inhibitory factor (MIF; 100 ng/ml) during 1, 3, 5 and 7 days of treatment. Each data point represents mean ± standard deviation from three independent experiments; *P <0.05 versus aged. mRNA levels of (B) vascular endothelial growth factor (VEGF), (C) basic fibroblast growth factor (bFGF), (D) hepatocyte growth factor (HGF) and (E) insulin-like growth factor (IGF) analyzed by quantitative real-time PCR in the culture of young, aged and MIF-treated aged MSCs, under normal and hypoxic conditions. Each column represents mean ± standard deviation from three independent experiments; *P <0.05 versus aged. Relative concentration of (F) VEGF, (G) bFGF, (H) HGF and (I) IGF analyzed by enzyme-linked immunosorbent assay, in the culture medium of young, aged and MIF-treated aged MSCs under normal and hypoxic conditions. Each column represents mean ± standard deviation from three independent experiments; *P <0.05 versus aged. (J) Representative distributions of propidium iodide (PI) and Annexin V staining from three FACScan flow cytometric analyses of apoptotic cells in normal and hypoxic conditions, in cultures of young, aged and MIF-treated (100 ng/ml added at the point of exposure to hypoxia and serum deprivation (hypoxia/SD) and maintained as such for 6 hours) MSC cultures: live (bottom left, Q-III), necrotic (top left, Q-I), early apoptotic (bottom right, Q-IV), late apoptotic (top right, Q-II). (K) Fold-change of apoptotic cells compared with corresponding control cells. Each column represents mean ± standard deviation from three independent experiments. *P <0.05 versus hypoxia/SD + aged, ^△ P <0.05 versus normal + aged, ^◇ P <0.05 versus normal + young.