title: Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.
abstract: Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.
authors.affiliation: Comparative Orthopedic Molecular Medicine and Applied Research Laboratories, Department of Veterinary Clinical Sciences, The Ohio State University, 601 Tharp Street, Columbus, Ohio 43210, USA. zachos.1@osu.edu
authors.forename: Terri A
authors.initials: TA
authors.lastname: Zachos
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Shields
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Bertone
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation; D054426:Chemokine CXCL2; D019743:Chemokines, CXC / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics*; D014161:Transduction, Genetic; D016212:Transforming Growth Factor beta / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D015854:Up-Regulation; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C525718:BMP2 protein, human; C525816:BMP6 protein, human; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D055418:Bone Morphogenetic Protein 6; D019485:Bone Morphogenetic Proteins; C499706:CXCL2 protein, human; D054426:Chemokine CXCL2; D019743:Chemokines, CXC; D016209:Interleukin-8; D016212:Transforming Growth Factor beta; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase