Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy

Abstract

Background Among the approximately 6.5 million fractures suffered in the United States every year, about 15% are difficult to heal. As yet, for most of these difficult cases there is no effective therapy. We have developed a mouse radial segmental defect as a model experimental system for testing the capacity of Genetically Engineered Pluripotent Mesenchymal Cells (GEPMC, C3H10T1/2 clone expressing rhBMP‐2), for gene delivery, engraftment, and induction of bone growth in regenerating bone. Methods Transfected GEPMC expressing rhBMP‐2 were further infected with a vector carrying the lacZ gene, that encodes for β‐galactosidase (β‐gal). In vitro levels of rhBMP‐2 expression and function were confirmed by immunohistochemistry, and bioassay. Differentiation was assayed using alkaline phosphatase staining. GEPMC were transplanted in vivo into a radial segmental defect. The main control groups included lacZ clones of WT‐C3H10T1/2‐LacZ, and CHO‐rhBMP‐2 cells. New bone formation was measured quantitatively via fluorescent labeling, X‐ray analysis and histomorphometry. Engrafted mesenchymal cells were localized in vivo by β‐gal expression, and double immunofluorescence. Results In vitro, GEPMC expressed rhBMP‐2, β‐gal and spontaneously differentiated into osteogenic cells expressing alkaline phosphatase. Detection of transplanted cells revealed engrafted cells that had differentiated into osteoblasts and co‐expressed β‐gal and rhBMP‐2. Analysis of new bone formation revealed that at fout to eight week post‐transplantation, GEPMS significantly enhanced segmental defect repair. Conclusions Our study shows that cell‐mediated gene transfer can be utilized for growth factor delivery to signaling receptors of transplanted cells (autocrine effect) and host mesenchymal cells (paracrine effect) suggesting the ability of GEPMC to engraft, differentiate, and stimulate bone growth. We suggest that our approach should lead to the designing of mesenchymal stem cell based gene therapy strategies for bone lesions as well as other tissues. Copyright © 1999 John Wiley & Sons, Ltd.

Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type

“…11 Our study also indicates that transduced human cells do not differentiate along a chondro-osseous pathway and do not incorporate into the newly formed cartilage or bone. This conclusion differs from those of several studies, 4,8,9 although orthotopic rather than heterotopic bone was analyzed. The fact that several cell types can be transduced and elicit bone formation also argues against Adenovirus BMP2-induced bone formation Z Gugala et al a major contribution of the transduced cells as constituents of these new tissues.…”

“…Musgrave et al 10 demonstrated a diverse response in a heterotopic bone formation model, depending on the cell type used for Ad5-BMP2 transduction and intramuscular implantation. Osteogenic cells that were either genetically engineered 8 or transduced 9,18 to express BMP2 have been shown to induce orthotopic bone formation. However, in these studies the cells were delivered using a carrier.…”

“…4,8,9,18 In our study, cells were injected without carrier or scaffold, whereas in all other studies collagen carriers were utilized. 4,8,9,18 Presumably, the carrier immobilizes the cells, decreasing their clearance rate. Additionally, the carrier may also retain high amounts of BMP2, forcing bone formation within the carrier, which can result in juxtaposition of the transduced cells and the new bone.…”

“…In addition to expressing BMP2, such cells may directly participate in bone formation as targets of BMP2 activity. 9 Specific cell types with various osteogenic potentials have been examined by Musgrave et al 10 for their ability to form heterotopic bone following ex vivo transduction with Ad5-BMP2. Among the cells studied, primary mesenchymal stem cells (MSCs) and primary articular chondrocytes did not induce bone detectable radiographically, whereas primary muscle-derived cells were capable of forming bone in vivo.…”

Spine fusion by gene therapy

“…The Ad-hBMP-2 construct when transduced into murine stromal, 35 C2C12 myoblasts, 36 primary muscle-derived cells, 37,38 or mesenchymal progenitor cells 39,40 induced bone when injected intramuscularly in rodent muscle beds, rodent critical sized skull defects, 41,42 or in a mouse radial segmental defect models. 43,44 A similar ex vivo approach has been successful using bone marrow cells in rat femoral defects, 45 but unsuccessful for spine fusion in rabbits. 46 A similar ex vivo gene therapy followed by cell transplantation exists with other BMPs.…”