FIG. 4.

Enhanced therapeutic potency of S1P primed hCB-MSCs. (A) hCB-MSCs were prestimulated with the indicated dose of S1P or LL-37. After 1 day, 60 cells were seeded into six-well culture plates and cultured for 14 days in complete medium. The number of colonies was counted and are presented as the mean±SD (n≥8); *P<0.05, **P<0.01, compared to cells in medium alone (one-way ANOVA with Bonferroni post-test). Representative stained colonies of adherent cells are shown in the right panel. (B) A schematic diagram for the in vitro anti-inflammatory activity assay (left panel). Quantification of tumor necrosis factor-α protein secreted from a murine alveolar macrophage cell line stimulated with lipopolysaccharides (LPS) for 5 h in the absence or presence of conditioned medium (CM) harvested from the indicated cells. Data are presented as mean±SD (n=9); *P<0.05, **P<0.01 compared to media alone (one-way ANOVA with Bonferroni post-test). S1P-MSC indicates hCB-MSC primed with 0.2 μM S1P for 1 day. (C, E) RQ-PCR analysis of the inflammation- (C) and angiogenesis-related (E) genes in hCB-MSCs after treatment with 0.2 μM S1P or 2.5 μg/mL LL-37 for 24 h. The relative expression level of the indicated genes is represented as the fold change compared to the value of MSCs cultured without priming (N.T.) and are shown as mean±standard error of mean (SEM, n=5); **P<0.01 compared to N.T. (one-way ANOVA with Bonferroni post-test). (D) Cell proliferation analysis of human umbilical vein endothelial cells at the indicated days after treatment of CM harvested from the indicated cells. Fold differences relative to IMR90 at 1 day are shown as the mean±S.D. (n=10), (*P<0.05, **P<0.01 compared to N.T.).