Figure 3

Addition of SDF-1α to the preconditioning protocol induces simultaneous upregulation of Col7a1, Tsg-6, and Cxcr4 mRNA expression. Mesenchymal stem cells (MSCs) were treated with 15 ng/ml transforming growth factor beta +30 ng/ml tumor necrosis factor alpha for 48 hours. At 47 hours, cells were exposed to 30 ng/ml stromal cell-derived factor 1-alpha (SDF-1α) for 1 hour. (a) Quantitative polymerase chain reaction (qPCR) was performed for Col7a1, Tsg-6, and Cxcr4 expression in treated cells relative to untreated MSCs. qPCR values were normalized against endogenous glyceraldehyde 3-phosphate dehydrogenase expression, and experiments were run in triplicate and across two experimental groups. (b) Flow cytometry was performed to assess cell surface CXCR4 expression in treated versus untreated cells. (c) Chemotaxis assay results of treated versus untreated cells: 50,000 GFP-expressing cells were placed in each top well, while increasing SDF-1α gradients were used in the bottom wells. For blocking controls, treated and untreated cells were incubated in presence of 100 μg/ml AMD3100 for 1 hour and exposed to a 90 ng/ml SDF-1α concentration gradient during the assay. Experiments were run in duplicate. (d) Representative fluorescent microscopy images of the chemotaxis membrane following the assay. Data presented as mean ± standard deviation. *P <0.05 by Student’s t test.