wnt signaling enhances proliferation and osteogenesis and suppresses adipogenesis of bone marrow-derived mesenchymal stem cell

Mesenchymal stem cells are multipotent cells that can differentiate to mesodermal lineages such as bone, fat, tendon and cartilage. To this end, these cells have been isolated from various tissues, but the main source is bone marrow. Currently, these cells are one of the most promising candidate for gene therapy, cell therapy and tissue engineering. The main problem in usage of these cells for these goals is reduction of proliferation and differentiation potential, and increasing of apoptosis in vitro and after transplantation in vivo. Wnt signaling pathway is one of the important signaling pathways which regulates proliferation, differentiation, polarity and apoptosis in all animal cells. It is well known that lithium has ability to inhibit GSK-3β, thus activate the function of wnt signaling pathway. Therefore, in the present study, we focuses on the effects of lithium on proliferation, apoptosis and differentiation of MSCs. MSCs were isolated from rat bone marrow and cultured under different lithium concentrations. Proliferation of these cells under different lithium concentrations considered by colony assay, population doubling and growth curve. Viability and survival of these cells under mentioned concentrations in third passage was studied by MTT and flow cytometry. The cells that have been cultured until secondary passage under 2mM, 5mM and 10mM concentration of lithium were selected for apoptosis assay. In these cells apoptosis were induced by use of simultaneously treatment of TNF-α and serum deprivation. As induction of apoptosis, these cells were treated with lithium and progression and occurring of apoptosis was studied by acridin orange-ethidium bromide staining after 24, 48 and 72 h. To study the effects of GSK-3β inhibition in osteogenesis and adipogenesis-induced by differentiation mediums, the cells also treated with lithium. Osteogenesis level was evaluated by gene expression of ALP and osteocalcin and amount of mineral matrix precipitation under different concentration of lithium. The effects of GSK-3β inhibition on adipogenesis was studied by light microscopy. The most number of expanded-colonies in primary cultured cells observed in 5mM lithium and these colonies were larger than other treatment and control groups. In addition, growth curve and population doubling number assay showed that 5mM lithium is the best appropriative concentration for proliferation. Also, efficient dose for cell viability under non-proliferative condition is 5mM lithium. Staining by acridin orange-ethidium bromide showed that GSK-3β inhibition by lithium could be suppressed apoptosis induction and controlled its progression. Increasing of gene expression of ALP, Osteocalcin and mineral matrix precipitation under lithium treatment indicated that GSK-3β have an inhibitory role in osteogenesis. Light microscopy showed that the number of adipocyts and the size of fatty granule is considerably decreased in lithium groups with respect to control group. Together, our results showed that lithium by inhibiting of GSK-3β causes an increase in MSCs viability, proliferation and decrease the apoptosis induced by TNF-α and serum deprivation. Also osteogenesis and adipogenesis are stimulated and inhibited respectively.

کلیدواژه ها:

Mesenchymal stem cells ، Wnt signalling ، Lithium chloride ، apoptosis ، proliferation ، osteogenesis ، adipogenesis