SUMMARY DEVELOPMENT AND DISEASE BMP signaling is required for septation of the outflow tract of the mammalian heart

No abstract

The aim of this study was to evaluate the use of transplantation of genetically modified allogeneic cells as a method to induce bone formation. In this study, we infected a murine osteoprogenitor cell line with a retroviral vector containing the human bone morphogenic protein 2 (BMP2) gene. Transduced cells exhibited more alkaline phosphatase activity than cells treated with any of the tested doses of recombinant human BMP2 protein (rhBMP2). The transduced cells were suspended in a collagen solution and injected into the quadriceps muscle in immunocompetent outbred mice. Radiographic and histological examinations demonstrate abundant ectopic bone formation in 85% of the transplanted animals (n = 13). PCR and Southern blot analysis for the puromycin resistance gene revealed that the transplanted cells were detectable for up to 1 week, but not at later time points. None of the animals developed tumors. Our results suggest that allogeneic BMP2-expressing transduced cells may have therapeutic potential for enhancing new bone formation. This model also provides a simple, inexpensive, and sensitive assay for evaluating in vivo the osteoinductive potentials of secreted proteins without the requirement of protein purification or the use of immunodeficient animals.

Ex vivo gene therapy using stem cells transduced with viral vectors is a useful method for delivering a therapeutic protein to augment bone repair in animal models. However, the duration of cell-mediated protein production and the fate of the transduced cells are unknown. We constructed an adenoviral vector encoding Myc epitope tagged bone morphogenetic protein (BMP)-2 gene (AdBMP-2 Myc ). Rat bone marrow cells transduced with this vector produced biologically active BMP-2 protein, which was confirmed by Western blot analysis and alkaline phosphatase assay. Implantation of bone marrow cells infected ex vivo with AdBMP-2 Myc caused orthotopic bone formation in mouse hindlimbs and bony union of critical-sized mouse radial defects. Immunohistochemical analysis revealed that rBMCs expressed Myc epitope-tagged BMP-2 protein for 14 days in vivo and became incorporated in the endochondral fracture callus. This novel adenovirus encoding for epitope-tagged BMP-2 can be used for immunohistochemical tracking of transduced cells in ex vivo gene therapy for bone repair.Treatment of bone defects secondary to trauma, tumor, or infection often requires bone grafting. Bone grafting can be accomplished with autogenous bone graft, allograft bone from cadaveric donors, demineralized bone matrices, calcium-phosphate ceramics, or recombinant proteins. Autogenous bone graft from the iliac crest often is considered the first choice of bone-graft materials, but the supply is limited. In addition, harvesting autogenous bone graft can be associated with morbidity such as donor-site pain, nerve damage, and bleeding. 1,5,18 Increased attention has focused on using recombinant proteins such as BMPs to induce bone repair. 14,19 However, supraphysiologic doses of recombinant BMPs are required to induce bone formation in clinical cases. 7,14,34 There is also concern that one dose of exogenous protein will not be sufficient to induce an adequate osteoinductive response in patients with large bone defects or with significant soft tissue injury. 10 One potential alternative to direct recombinant protein delivery is to develop a biologic cellular delivery vehicle via gene therapy to enhance bone formation. 8 Ex vivo gene therapy has been used successfully in various preclinical animal models to promote bone repair. Critical-sized femoral and calvarial defects have healed successfully using ex vivo gene therapy strategies with adenoviral and retroviral vectors. 9,16,20,21 The duration of in vivo protein production and the fate of the infected cells in vivo still need to be defined. Do the transduced cells just act as cellular delivery vehicles or do they actively participate in the bone repair process? Bone marrow cells, adipose-derived stem cells, and muscle-derived stem cells, all of which can differentiate into bone under the appropriate stimuli, have been used as cellular delivery vehicles in ex vivo gene therapy strategies. 12,15,17,21,28,30 A potential advantage of using osteoprogenitor cells is that these transduced cells not only pr...

To catalog factors that may contribute to the completion of myogenesis, we have been looking for molecular differences between BC3H1 and C2C12 cells. Cells of the BC3H1 tumor line, though myogenic, are nonfusing, and withdraw from the cell cycle only reversibly, whereas cells of the C2C12 line fuse, differentiate terminally, and express several muscle-specific gene products that BC3H1 cells do not. Relative to C2C12 cells, BC3H1 cells underaccumulated cyclin-dependent kinase inhibitor p21 and underaccumulated transcripts for p21, GADD45, CDO, decorin, osteopontin, H19, fibronectin, and thrombospondin-1 (tsp-1). Levels of accumulation of H19, tsp-1, and larger isoforms of fibronectin messenger ribonucleic acid (mRNA) were found to increase in response to expression of myogenic regulatory factors as shown by their accumulation in differentiated myogenically converted 10T1/2 cells but not in 10T1/2 fibroblasts. BC3H1s accumulated a temperature-insensitive, geldanamycin-sensitive, misfolded form of p53 incapable of transactivating a p53 responsive reporter, consistent with underexpression of p21, GADD45, and tsp-1. BC3H1 and C2C12 cells were similar with respect to upregulation of p27 protein, downregulation of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein, upregulation of retinoblastoma (Rb) mRNA, and nuclear localization of hypophosphorylated Rb. Cells of both lines expressed the muscle-specific 1b isoform of MEF2D. Although nonfusing in the short term, after more than 18 d in differentiation medium, some cultures of BC3H1 cells formed viable multinucleated cells in which the nuclei did not reinitiate synthesis of DNA in response to serum. Our findings suggest participation of tsp-1 and specific isoforms of fibronectin in myogenesis and suggest additional avenues of research in myogenesis and oncogenesis.