After incubation, the reaction contents were loaded on the 0. 8% agarose gel and run for 2 to 3 hr at 5 to 10 V/cm. The sample topoisomerase activity was then relatively selleck determined by the percentage of supercoiled plasmid. Statistical analysis All experiments included at least 3 biological repeats and all values were presented as mean standard devi ation. Statistical comparisons were analyzed with the two tailed Students t test or one way ANOVA with Tukey multiple comparison. A P value less than 0. 05 was consid ered statistically significant. Results The expression of HPSE1 by mouse BM MSCs and the enzymatic inhibition by OGT2115 To test our hypothesis that cell autonomous heparanase participated in the maintenance of stem cell niche, we first demonstrated that mouse BM MSCs express HPSE1.

RT PCR and immunocytochemistry showed that isolated BM MSCs consistently express HPSE1 at mRNA and protein levels. Heparanase is trans lated Inhibitors,Modulators,Libraries as a pro enzyme and requires to be processed into a smaller molecule to be enzymatically active. To clarify whether heparanase expressed by isolated BM Inhibitors,Modulators,Libraries MSCs is enzymatically active, cell lysates were harvested for west ern blot. Western blot detected both Inhibitors,Modulators,Libraries intact heparanase and activated heparanase indicating that the BM MSCs not only can express HPSE1 but also can activate it. To assess the role of this enzyme in BM MSCs, we exploited the small molecule heparanase inhibitor OGT2115 to block the enzymatic activity of heparanase.

Dot blot of cell extracts with the addition of OGT2115 showed significantly stronger reactivity against complete heparan sulfate chain content antibody Inhibitors,Modulators,Libraries when com pared to the vehicle control indicating that HPSE1 Inhibitors,Modulators,Libraries enzymatic activity in BM MSCs was efficiently inhibited by OGT2115. Inhibition of HPSE did not affect molecular phenotypes and osteogenic differentiation To assess whether the inhibition of HPSE alters extrinsic signals and in turn affects stem cell property, we analyzed a panel of surface markers of mouse MSCs. Mouse BM MSCs were positive for MSCs markers Sca 1, CD73, and CD105, while negative for the hematopoietic cell marker, CD45, BI 6727 and for the endothelial cell marker, CD31. These expression profiles remain identi cal not only through serial passages at passage 2, passage 4 and passage 7, but also in the presence of OGT2115 indicating that the inhibition of HPSE activity does not alter the identity of BM MSCs. Previous studies indicated that environmental heparan sulfate degrading activity enhanced the osteogenic differ entiation of BM MSCs. We therefore assessed the potential role of HPSE1 in the osteogenic differenti ation of mouse BM MSCs.