Engineered pluripotent mesenchymal cells integrate and differentiate in regenerating bone: a novel cell-mediated gene therapy.
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 beta-galactosidase (beta-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 beta-gal expression, and double immunofluorescence.


RESULTS
In vitro, GEPMC expressed rhBMP-2, beta-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 beta-gal and rhBMP-2. Analysis of new bone formation revealed that at four 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.
Gazit
D
D
Molecular Pathology Laboratory, Hebrew University-Hadassah Faculty of Dental Medicine, Jerusalem, Israel. dgaz@cc.huji.ac.il
Turgeman
G
G
Kelley
P
P
Wang
E
E
Jalenak
M
M
Zilberman
Y
Y
Moutsatsos
I
I
D000818:Animals; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D001842:Bone and Bones / Q000166:cytology / Q000502:physiology*; D016466:CHO Cells; D002454:Cell Differentiation; D002460:Cell Line; D017690:Cell Transplantation; D006224:Cricetinae; D005260:Female; D017102:Fracture Healing / Q000235:genetics / Q000502:physiology; D005818:Genetic Engineering; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D008954:Models, Biological; D011885:Radius Fractures / Q000473:pathology / Q000503:physiopathology / Q000628:therapy; D012038:Regeneration / Q000235:genetics* / Q000502:physiology*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta
C525718:BMP2 protein, human; C525719:Bmp2 protein, mouse; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta