title: Enhanced in vitro chondrogenesis of primary mesenchymal stem cells by combined gene transfer.
abstract: Because articular cartilage has a poor regeneration capacity, numerous cell-based approaches to therapy are currently being explored. The present study involved the use of gene transfer as a means to provide sustained delivery of chondrogenic proteins to primary mesenchymal stem cells (MSCs). In previous work, we found that adenoviral-mediated gene transfer of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2), but not insulin-like growth factor 1 (IGF-1), could be used to induce chondrogenic differentiation of MSCs in an aggregate culture system. In the present study, we examined the effects on chondrogenesis of these transgenes when delivered in combination. Cultures of bone marrow-derived MSCs were infected with 2.5 x 10(2) or 2.5 x 10(3) viral particles/cell of each adenoviral vector individually, or in combination, seeded into aggregates, and cultured for 3 weeks in a defined serum-free medium. Levels of transgene product in the medium were initially high, approximately 100 ng/mL TGF-beta1, 120 ng/mL BMP-2, and 80 ng/mL IGF-1 at day 3, and declined thereafter. We found that co-expression of IGF-1 and TGF-beta1, BMP-2, or both at low doses resulted in larger aggregates, higher levels of glycosaminoglycan synthesis, stronger staining for proteoglycans and collagen type II and X, and greater expression of cartilage-specific marker genes than with either transgene alone. Gene-induced chondrogenesis of MSCs using multiple genes that act synergistically may enable the administration of reduced viral doses in vivo and could be of considerable benefit for the development of cell-based therapies for cartilage repair.
authors.affiliation: Harvard Medical School, Center for Molecular Orthopaedics, Boston, Massachusetts, USA. a-steinert.klh@uni-wuerzburg.de
authors.forename: Andre F
authors.initials: AF
authors.lastname: Steinert
authors.forename: Glyn D
authors.initials: GD
authors.lastname: Palmer
authors.forename: Carmencita
authors.initials: C
authors.lastname: Pilapil
authors.forename: Ulrich
authors.initials: U
authors.lastname: Nöth
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Evans
authors.forename: Steven C
authors.initials: SC
authors.lastname: Ghivizzani
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D055396:Bone Morphogenetic Protein 2 / Q000235:genetics / Q000502:physiology; D002417:Cattle; D002449:Cell Aggregation; D002454:Cell Differentiation; D020219:Chondrogenesis / Q000235:genetics* / Q000502:physiology*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006025:Glycosaminoglycans / Q000096:biosynthesis; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000502:physiology; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000502:physiology
chemical_list: D055396:Bone Morphogenetic Protein 2; D017931:DNA Primers; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D053773:Transforming Growth Factor beta1; D007334:Insulin-Like Growth Factor I; D004247:DNA