Several gene therapy approaches were developed aiming to achieve bone tissue regeneration, based on viral gene delivery of osteogenic factors. The limitations of viral-based approaches have been extensively reported mostly dealing with the humoral immune response generated against the viral proteins. An alternative approach is the use of non-viral gene therapy, which is considered to be safer but lacks the high efficiency of gene transduction available with viral vectors. Electroporation might be an efficient physical nonviral method to deliver DNA molecules into stem cells. We have shown that cell-mediated gene therapy using virally trasduced mesenchymal stem cells can efficiently regenerate non-union bone defects. Therefore, we hypothesized that in vivo bone formation could be achieved using human Mesenchymal Stem Cells (hMSCs) nonvirally transfected with BMP genes. HMSCs were isolated from bone marrow samples and transfected using a Nucleofector (Amaxa Biosystems), a novel electroporation-based technique. We first utilized the GFP and Luciferase reporter genes in order to optimize the viability of hMSCs, the efficiency of DNA transfer and the duration of transgene expression following gene transfer. FACS analysis, quantitative PCR and Luciferase biochemical assay were used for the analysis. Moreover, we have manipulated hMSCs to G0/G1 cell cycle phase, prior to transfection, in order to determine whether it would increase transfection efficiency. In the second stage we utilized the osteogenic genes, BMP-2,-6 and-9, in order to induce osteogenic differentiation. Transfected hMSCs, embedded in Fibrin gel carrier (Baxter) were also injected into the thigh muscle or in the lumbar region, of NOD/SCID mice. We were able to monitor injected cell survival in vivo by transfecting the cells with a Luciferase plasmid, and noninvasively detecting them quantitatively, using a CCCD system (B). The sites of injection were scanned in vivo and ex vivo, using uCT system. Anti human mitochondria assay was used to demonstrate the integration of the engineered cells in bone formation (C). Our in vitro results demonstrated 53% cell viability and up to 70% transfection efficiency. The transfection efficiency was increased when 98% of the cells were in Go/G1 phase prior to transfection, and transgene expression lasted at least 14 days in vitro. Osteogenic differentiation was demonstrated in hMSCs transfected nonvirally with different BMPs (A). In vivo bone formation was evident in the injection sites (D, E), and the nonvirally transfected hMSCs were able to induce spinal fusion in the lumbar region. We conclude that hMSCs can be effectively transfected using this nonviral novel technique. These findings could pave the way to a safe and efficient gene therapy for human bone regeneration Figure 1.