Effect of metformin on osteogenic and adipogenic differentiation of rat mesenchymal stem cells

Abstract

Objective To investigate the effect of metformin on osteogenic and adipogenic differentiation of primary bone marrow mesenchymal stem cells. Methods Bone marrow mesenchymal stem cells were isolated and purified from the rat bone marrow by adherent method, the cells from passage 3-4 were used in our experiments. At 90% confluence, the medium was removed and the cells were cultured in osteogenic medium with or without 10 μmol/L metformin, medium was changed every two or three days. At days 7, 14, and 21, the cells were collected, osteogenic differentiation was evaluated by measuring Runt-related transcription factor 2, alkaline phosphatase, collagen type Ⅰ and osteocalcin using real-time PCR; ALP activity was also evaluated by p-nitrophenyl phosphate as the substrate and normalized to total protein content; At day 21, mineralization nodules in the extracellular matrix of bone marrow mesenchymal stem cells were stained using Alizarin red staining. Differentiation of adipocytes were induced in adipogenic medium for 14 days with or without 10 μmol/L metformin, then the cells were collected for detecting the transcription activity of PPARγ and CAAT/enhanced-binding protein α, lipid droplets in adipocytes were measured by oil red O staining. Results In the metformin group, alkaline phosphatase activity was higher, osteoblast markers were up-regulated(all P＜0.05), and the calcium nodules were more, while the adipocyte markers are down-regulated by about 70%, and lipid droplets were less than the group without metformin. Conclusion Metformin can effectively promote the differentiation of bone marrow mesenchymal stem cells to osteoblasts, while inhibit the differentiation to adipocytes. Key words: Metformin; Bone marrow mesenchymal stem cells; Osteoblasts; Adipocytes