The effect of purmorphamine and sirolimus on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

Abstract

Small molecules have been introduced as less expensive biologically active compounds that can regulate different developmental phenomena. Purmorphamine and sirolimus are two small molecules that, according to some studies, possess certain osteomodulatory effects. This study was set out to highlight the appropriate dose and response time of these small molecules on enhancement of osteogenesis in human bone marrow-derived mesenchymal stem cells from early to mid and late stages of differentiation. Alkaline phosphatase activity, matrix mineralization and expression of osteoblast genes were quantitatively assessed in vitro. For the in vivo study, we transplanted stem cell-based constructs subcutaneously into rats, and treated them daily with the most promising doses of the small molecule. The constructs were analyzed by real-time PCR and histological staining. Our results showed that Sirolimus reduced osteogenic differentiation of mesenchymal stem cells by decreasing alkaline phosphatase activity at dose of 100nM after 14 days and mineralization of the matrix at 14 and 21 days post-induction. Purmorphamine induced up-regulation of alkaline phosphatase activity and expression of RUNX-2 at day 14. Up-regulation of osteocalcin was detected at the 3 and 5μM doses of purmorphamine on day 14 post-induction. Matrix mineralization remained unchanged in the presence or absence of purmorphamine. This dose of small molecule also accelerated expression of Alkaline phosphatase transcripts in vivo. In conclusion, sirolimus had an inhibitory effect on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells; while purmorphamine, particularly at a dose of 3μM, showed a promotive effect in vitro and in vivo.