240. Lenti-BMP2/GFP Gene Therapy Enhance the Chondrogenic and Osteogenic Differentiation of Human Muscle Derived Stem Cells In Vitro

Human muscle derived stem cells (hMDSCs) have been shown to be multipotent in vitro and can repair critical size bone defects as efficiently as human bone marrow stem cells, but require BMPs. In this study, we constructed a new lenti-BMP2/GFP vector and tested its ability to promote in vitro chondrogenesis and osteogenesis of the hMDSCs. Material and methods: 1. Lenti-BMP2/GFP was made by inserting human BMP2 gene up-stream of an eGFP tag gene in a Lenti-viral vector with a CMV promoter. 2. hMDSCs were isolated from skeletal muscle via the preplate technique. All 6 populations were transduced with the Lenti-BMP2/GFP virus, expanded and sorted via FACS based on GFP fluorescence. BMP2 secretion was measured using an R&D ELISA kit. 3. Osteogenesis:3D pellet cultures were used for osteogenic differentiation of non-transduced and Lenti-BMP2/GFP transduced hMDSCs. After 4 weeks, the cell pellets were fixed and scanned using VivaCT40 to detect mineralization. The pellets were embedded in NEG medium and snap frozen in liquid nitrogen, sectioned and von Kossa stained. Osteogenic differentiation was detected by staining for osteocalcin. 4. Chondrogenesis was performed on non-transduced and Lenti-BMP2/GFP transduced hMDSCs using a 3D pellet culture method. Cell pellets were cultured for 24 days in chondrogenic medium, embedded in freezing medium and cryosectioned. Alcian blue staining and Col2A1 immunohistochemistry was also performed. Raman Spectroscopy was used to quantify chondroitin sulphate and collagen. Results: 1. Lenti-BMP2/GFP transduction enhanced osteogenesis of the hMDSCs. BMP2 secretion levels of the sorted lenti-BMP2/GFP transduced cells are showed in Fig. 1A. MicroCT results of the lenti-BMP2/GFP transduced cells showed they had an increase in mineralized pellet volume (Fig. 1B, C). Von Kossa staining indicated that the non-transduced hMDSCs had variable brown-black mineralization while the lenti-BMP2/GFP transduced cells all showed stronger mineralization (Fig. 1D). Osteocalcin staining showed lenti-BMP2/GFP transduction enhanced osteogenic differentiation of the hMDSCs (Fig. 1E). 2. Lenti-BMP2/GFP transduction enhanced chondrogenesis of the hMDSCs. Alcian blue staining showed a significantly bluer chondrogenic matrix in the Lenti-BMP2/GFP transduced cell pellets compared to non-transduced hMDSCs (Fig2A and 2B). Raman Spectroscopy indicated the Lenti-BMP2/GFP transduced hMDSC exhibited higher amounts of chondroitin sulphate and collagen (Fig. 2C, D). Col2A1 staining showed more Col2A1 in the lenti-BMP2/GFP transduced hMDSCs compared to non-transduced hMDSCs (Fig. 2E). Conclusion: We found that all the Lenti-BMP2/GFP transduced hMDSCs showed enhanced osteogenesis and chonrogenesis compared to non-transduced cells. This in vitro study lays the foundation for using Lenti-BMP2/GFP gene transfer to repair bone and cartilage defects.