Fig. 5: Secreted OPN from MSCs acts as a chemotactic factor for MSCs under elevated extracellular Ca²⁺ conditions.

a Extracellular Ca²⁺ itself does not act as a cellular chemoattractant. C3H10T1/2 cells were used in Transwell assay with standard medium or 6 mM Ca²⁺ medium in the lower chamber. Cells that migrated to the lower side of the well for the indicated time were fixed and stained. b and c Conditioned medium (CM) from C3H10T1/2 cells cultured under the elevated extracellular Ca²⁺ condition induces cell migration. b Migration of C3H10T1/2 cells for 6 h to the lower chamber containing the indicated medium. Standard medium supplemented with the indicated concentration of CaCl₂ (fresh medium, FM) was used as a control. c Migration of BM-MSCs for 6 h to the lower chamber containing in the indicated medium. d Migration inducible ability of CM is abolished following inhibition of new protein synthesis. C3H10T1/2 cells were seeded with or without 5 μg/mL of cycloheximide (CHX) in the upper chamber. Cells that migrated to the lower side of the well for 24 h were fixed and stained. e-g Extracellular OPN enhances migration of MSCs. e Depletion of OPN in the CM by using neutralizing antibodies abolishes the elevated extracellular Ca²⁺-induced cell migration. The indicated CM was pre-incubated with an OPN neutralizing antibody for 1 h and used as the lower chamber medium. C3H10T1/2 cells that migrated to the lower side of the well for 6 h were fixed and stained. f Migration of C3H10T1/2 cells for 6 h to the lower chamber containing the indicated amount of recombinant OPN in standard medium. Number of migrated cells expressed as fold increases relative to control (e and f). g Migration of BM-MSCs for 4 h to the lower chamber containing 0.1% BSA (control), 20 ng/mL TGFβ1, 20 ng/mL OPN, or 10 ng/mL FGF2. Number of migrated cells per field of view (FV, x50 magnification) is indicated (a, b, d, and g). Number of migrated cells expressed as the fold increases relative to control (c, e, and f). *p < 0.05; **p < 0.01; ***p < 0.001 vs. indicated group. p < 0.001 (a–e). *p < 0.05; **p < 0.01; ***p < 0.001 vs. first bar (f and g)