Fig. 4

Monocyte differentiation and attraction in the presence of dental MSCs. Monocytes alone (Mo) or in the presence of green fluorescent protein (GFP)-mesenchymal stem cells (MSCs; light grey) and hypoxia-inducible factor (HIF)-MSCs (dark grey) were cultured with rhGM-CSF to induce macrophage differentiation. After 7 days, the expression of CD14 and CD163 was determined by flow cytometry. A representative experiment (a) and mean ± SD of three independent experiments (b) are shown. MSCs were excluded from the analysis by CD90 expression. c After 7 days of culture, LPS was added for 24 h and supernatants were assayed for tumor necrosis factor (TNF) alpha and interleukin (IL)-10 production. Data are presented as mean ± SD of three independent experiments. One-way ANOVA, *p ≤ 0.05, GFP-MSCs and HIF-MSCs versus no MSCs. d, e MSCs were cultured until 80% confluence, then fresh medium alone (Med) or medium supplemented with IFN-gamma (IFNγ) was added. After 15 h, cells and supernatants were harvested. Transcript (d) and protein levels (e) for CCL2 were determined by quantitative PCR and CBA, respectively. GNB2L1 was used as endogenous control. Mean ± SD of four samples pooled from two independent experiments is shown. Student t test, *p ≤ 0.05, MSCs vs HIF-MSCs). f Transwell (8 μm) cultures were performed as stated in the Methods section. After 14 h, migrated cells (present at the lower chamber) were collected and stained for CD14. MSCs were excluded from the analysis by CD90 expression and monocytes were gated according to forward/side scatter characteristics and expression of CD14, and counted in a FACSCalibur flow cytometer