Macrophage-Associated Mesenchymal Stem Cells Figure 6. Macrophage-secreted soluble factors induce MSC migration by activating the c-Jun NH2-terminal kinase (JNK) signaling pathway. MSCs were pre-incubated for 1 hour in control medium or control medium containing 25 mM ERK or p38 inhibitor. Neither ERK nor p38 inhibition influenced MSC migration in response to dU937 CM (A, B). MSCs were incubated in 25 mM JNK inhibitor for 1 hour prior to migration assay. JNK inhibition decreased MSC migration by 55% (*p, 0.006, n = 3)(C, D). The MSCs were incubated with the inhibitors for 18 hours after which MSC viability was calculated using trypan blue staining. JNK, p38, and ERK inhibitors did not significantly decrease MSC viability (E). doi:10.1371/journal.pone.0035036.g006 Materials and Methods Primary Culture Human Macrophages Buffy coats were prepared by the New Brunswick Affiliated Hospitals Blood Center (UMDNJ, New Brunswick, NJ) from peripheral blood samples donated by healthy volunteers. Each sample contained an average of 50 mL of blood. Mononuclear cells were isolated using a BD CPT Vacutainer tubes (8 mL per tube) (BD, Franklin Lakes, NJ). The resulting mononuclear cells were re-suspended in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum (FBS)(F0926 - Sigma Aldrich, St. Louis, MO) and 1% penicillin-streptomycin. Cell density was measured using an automated cell counter and cells were plated at a density of 8.06105 cells/cm2. Cells were allowed to adhere overnight and the medium was changed the following day. The cells were incubated at 37uC/5% CO2 for 7 days to allow for differentiation of monocytes into macrophages, receiving fresh medium every 3 days. Differentiated monocyte- derived macrophages exhibited increased production of IL-6, IL- 8, and TNF-alpha when treated with lipopolysaccharide (LPS) (Figure S1A). In addition, they express low levels of CD204, which is increased by treatment with IL-4 and IL-13 (Figure S1B). Primary Culture Mesenchymal Stem Cells Human bone marrow-derived mesenchymal stem cells (MSCs) harvested from pooled normal human donors were purchased from Lonza (Lonza, Walkersville, MD). Cellular identity was verified by flow cytometry and as well as the ability to differentiate into osteogenic, chondrogenic and adipogenic lineages as previously described [52]. Cells expressed CD105, CD166, CD29, and CD44 and were negative for CD14, CD34 and CD45. MSCs from two separate lots were used in this study. MSCs were expanded in Mesenchymal Stem Cell Growth Medium (Lonza, Walkersville, MD). For experiments using MSCs, cells were cultured in MEM-alpha medium (Invitrogen, Carlsbad, CA) containing 10% FBS and 1% penicillin-streptomycin. Fresh medium was added every 3–4 days and cultures were split to a lower density, using 1:1 trypsin-EDTA 0.25% (2.5g/L trypsin and 0.2 g/L EDTA 4Na in Hanks’ Balanced Salt Solution)(Sigma- Aldrich, St. Louis, MO):phosphate-buffered saline (PBS), once they achieved 80% confluence. All experiments were conducted with passage 3 cells. Differentiation of U937 Cells to dU937 Macrophages U937 cells (ATCC, Manassas, VA) were plated at the desired density in RPMI medium containing 10% FBS, 1% penicillin- PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35036