Macrophage-Associated Mesenchymal Stem Cells Figure 3. Macrophages induce MSC migration through the release of soluble factors. To determine the chemotactic potential of macrophages for MSCs the CM from primary culture macrophages and the dU937 macrophage cell line was harvested and used as the stimulus in a standard Boyden Chamber migration assay. Following an 18-hour migration, MSCs show increased motility and migration towards macrophage-like CM and primary macrophage CM when compared with control medium. Compared to control levels, the number of migrating MSCs is 5.7-fold greater towards dU937 CM (*p,0.003, n = 3) and 5.3-fold greater towards primary macrophage CM (**p,0.01, n = 3), respectively (A). A separate experiment was conducted to characterize the ability of dU937 macrophages alto induce MSC chemotaxis. When compared to control, the soluble factors secreted by the dU937 macrophage cell line induce an 8.7-fold increase in MSC migration while U937-secreted factors increased MSC migration 2.9-fold (B). dU937 macrophage cells induce a significantly higher amount of MSC migration than the undifferentiated cells (*p,0.03, n = 3). doi:10.1371/journal.pone.0035036.g003 Macrophage-secreted Soluble Factors Induce Phosphorylation of JNK1/2/3 and c-Jun In order to verify that JNK was activated in MSCs treated with dU937 CM we determined the levels of phosphorylated-JNK1/2/ 3 and downstream targets. Both phosphorylated-JNK1/2/3 (Figure 7A) and phosphorylated-c-Jun (Figure 7B) were increased after 30 minutes and returned to control levels by 2 hours. Phosphorylated-ATF-2 levels were not increased at 30 minutes, but rather decreased and remained reduced after 1 hour (Figure 7B) suggesting that MSC migration in response to macrophages may be mediated through c-Jun. Discussion MSCs were first isolated as adherent cells cultured from bone marrow and are non-phagocytic, exhibit a fibroblast-like pheno- type, and are able to differentiate into adipose, tendon, muscle, bone, and cartilage in vitro [20]. They can be activated and mobilized under the appropriate conditions and localize to solid tumors and injury sites [21–29]. Within the injury microenviron- ment, MSCs differentiate to a myofibroblast phenotype and participate in important aspects of wound healing including regulation of the immune response, inhibition of scarring and fibrosis, angiogenesis, and enhancement of mitosis of stem and progenitor cells during tissue repair [30–32]. Additionally, a growing body of evidence suggests that carcinoma-associated fibroblasts (CAF) are derived from MSCs [33]. MSCs differentiate into a CAF phenotype within the tumor microenvironment and support solid tumor growth and metastasis [33,34]. While the ability of MSCs to localize to sites of tissue damage and adopt specific phenotypes based on microenvironmental cues has been well established, the mechanisms responsible for these aspects of MSC biology are not well characterized. Macrophages are a major component of the inflammatory response in both injury and neoplasm, two cases in which MSC localization and integration is important. In this work we show that macrophage-secreted soluble factors alter the gene expression and cytokine secretion profiles of MSCs. Macrophages also induce MSC migration and influence the PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35036