size (Corning, New York, NY). Harvested CM was stored at 280uC overnight prior to analysis. All samples, including controls, contained FBS. The cytokine profile of each sample was analyzed using the Bio-Plex suspension array system (Bio-Rad Laboratories, Hercules, CA). The beads were mixed with the samples and incubated to react with the specific analytes. The addition of an analyte-specific biotinylated detection antibody and a fluorescently labeled reporter molecule, streptavidin-PE, completed the sand- wich immunoassay. The Bio-Plex array reader aligned the beads single file through a flow cell where two lasers individually excited the beads. Digital signal processors and the Bio-Plex Manager software recorded the fluorescent signals and quantitated the amount of analyte captured in each sample. All samples were assayed in triplicate. Conditioned medium samples displayed in Figure 2 were analyzed by the Cytokine Core Laboratory at the University of Maryland (Baltimore, MD). Transwell Migration Assay The assay was run as previously described [53]. A migratory stimulus (cells or CM) was plated into the well of a 24-well notched tissue culture plate (BD Falcon, San Jose, CA) in a total volume of 700 mL of culture medium. For primary macrophages, the cells were isolated from human peripheral blood samples (as described above) and plated directly into the 24-well plate and allowed to differentiate for 7–10 days prior to performing the experiment. For dU937 cells, U937 cells were plated in 20 nM phorbol 12- myristate 13-acetate (PMA) directly in the 24-well notched plate and allowed to differentiate for 4 days before performing the migration assay. When CM medium was used as a migratory stimulus, 700 mL was added into the 24-well notched plate and the migration assay was run immediately. Control base medium was RPMI+10% FBS, as used to culture macrophages. Once the stimulus was prepared, cell culture inserts (BD Falcon, San Jose, CA), containing an 8 mm pore size uncoated polyethylene membrane, were added to each well. 7.56103 human MSCs were added to the upper chamber of the cell culture insert in 500 mL of minimum essential medium (MEM)-a supplemented with 10% FBS and 1% penicillin-streptomycin. The tissue culture plates were then incubated at 37uC/5% CO2 for 18 hours. MSCs were allowed to migrate through the membrane towards the stimulus during this incubation. After 18 hours, the assay was terminated by aspirating the medium out of the top and bottom chambers, washing the insert with PBS, removing non-migrated cells out of the top chamber using a cotton swab, and fixing and staining the cells in the membrane with crystal violet. The number of MSCs that migrated was quantified manually using an inverted microscope. The human c-Jun N-terminal kinase (JNK) 1, 2, and 3 inhibitor, SP 600125 (Tocris Bioscience, Ellisville, MO), p38/MAPK inhibitor, SB 203580 (Tocris Bioscience, Ellisville, MO) and MAPK/ERK inhibitor, PD 98059 (Millipore, Temecula, CA) were used to analyze the role of these signaling systems in MSC migration. Small molecule inhibitors were added at a concentra- tion of 25 mM to the MSC culture medium for 1 hour prior to MSC harvesting and transwell migration study. In addition, References 1. Spaeth E, Klopp A, Dembinski J, Andreeff M, Marini F (2008) Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther 15: 730–738. 2. Ryu CH, Park SH, Park SA, Kim SM, Lim JY, et al. (2011) Gene Therapy of Intracranial Glioma Using Interleukin 12-Secreting Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells. Human gene therapy, 22(6) 733–43. Macrophage-Associated Mesenchymal Stem Cells inhibitors were added with the MSCs to the upper chamber of the transwell migration assay at the same concentration. RT-PCR RNA was isolated from cell cultures using the Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA). RNA quality was assessed by UV spectrophotometry, checking the RNA concentration and purity. cDNA was synthesized using the RT2 First Strand Kit (SABiosciences, Frederick, MD). Pathway-focused gene expression analyses were conducted on the samples using RT2 Profiler PCR Arrays (SABiosciences, Frederick, MD). The Human Inflamma- tory Cytokines and Receptors PCR array (PAHS-011 - SABios- ciences) profiled the expression of 84 key genes involved in mediating immune cascade reactions during inflammation. Data was analyzed with the SABiosciences RT2 Profiler Web-Based PCR Array Data Analysis software, which automatically performs the DDCt fold-change calculations from the uploaded raw threshold cycle data. Cell Viability To assess cell viability a Vi-Cell cell viability analyzer (Beckman Coulter) was used for cell counting. To test the viability of MSCs after macrophage association, MSCs cultured in macrophage CM were compared with MSCs cultured in control media (Figure S2). Statistical Analysis Student’s t-test was performed to compare results. A p value ,0.05 was considered statistically significant. Data are presented as mean 6 standard deviation. Supporting Information Figure S1 Macrophages respond to activating stimuli by increasing secretion of soluble factors and increasing expression of appropriate markers. Lipopolysaccharide (LPS) stimulation of the macrophage population induced increased secretion of multiple soluble factors including IL-6, TNF-a, CCL3, CCL5, and CXCL12 (A). Activation of macrophages with IL-4 and IL-13 increased the level of CD204 expression (B). These results suggest that the macrophages used in this study responded appropriately, as described in the literature, to multiple activation factors. (TIF) Figure S2 Cellular viability of MSCs is unaffected by stimulation with macrophage conditioned medium. Culturing of MSCs in dU937 conditioned medium did not alter cell viability when compared with MSCs cultured in control medium (RPMI supplemented with 10% FBS). (TIF) Author Contributions Conceived and designed the experiments: KA DB JG. Performed the experiments: KA. Analyzed the data: KA JG. Contributed reagents/ materials/analysis tools: JG DB. Wrote the paper: KA DB JG. 3. Bak XY, Dang HL, Yang J, Ye K, Lee EX, et al. (2011) Human Embryonic Stem Cell-derived Mesenchymal Stem Cells as Cellular Delivery Vehicles for Prodrug Gene Therapy of Glioblastoma. Human gene therapy 22(11): 1365–77. 4. Yang B, Wu X, Mao Y, Bao W, Gao L, et al. (2009) Dual-targeted antitumor effects against brainstem glioma by intravenous delivery of tumor necrosis factor- related, apoptosis-inducing, ligand-engineered human mesenchymal stem cells. Neurosurgery 65: 610–624. PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e35036