Adipose tissue contains multipotent mesenchymal stem cells (MSCs) that are able to differentiate into various tissues. Bone morphogenetic protein 2 (BMP2) is known as one of the key osteogenesis induction factors in MSCs. Recently, several new transcription factors that contribute to osteogenic differentiation have been reported, among them Runx2, Osterix, and Dlx5. We hypothesized that adipose-derived stromal cells (ASCs) could be induced to efficiently differentiate into osteocytes by the co-expression of the BMP2 and Runx2 genes. To prove this hypothesis, we constructed a bicistronic vector encoding the BMP2 and Runx2 genes linked to the 'self-cleaving' 2A peptide sequence. BMP2/Runx2-ASCs showed a gradual increase in alkaline phosphatase activity for two weeks. RT-PCR analysis and alizarin red staining revealed a high expression of osteogenesis-related markers (osteopontin, osteocalcin and collagen type I) and increased mineralization in BMP2/Runx2-ASCs compared to BMP2-ASCs. Six weeks after in vivo transplantation, BMP2/Runx2-ASCs also showed a significant increase in bone formation compared to ASCs and BMP2-ASCs. These findings demonstrate that the co-transfection of two osteogenic lineage-determining genes can enhance osteogenic differentiation of ASCs.
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