1095. Gene_Induced Chondrogenesis of Mesenchymal Stem Cells: A Comparative Analysis of Candidate cDNAs

Abstract

Articular cartilage is a highly specialized tissue that protects the ends of bones in diarthrodal joints. The cartilage extracellular matrix is maintained by chondrocytes that occupy the tissue at low density. Because articular cartilage is avascular, it has a poor capacity to regenerate; thus, the damage resulting from large injuries is considered permanent. Due to their ready availability and capacity to differentiate along numerous lineages, adult mesenchymal stem cells (MSCs) may be useful in the development of cell-based therapies for cartilage repair. In this regard, gene transfer to MSCs can be used to achieve sustained expression of specific protein factors capable of inducing chondrogenic differentiation. In this study, using adenoviral mediated gene transfer and bone marrow derived MSCs in an aggregate culture system, we compared the relative condroinductive capacity of several cDNAs whose protein products have been associated with the induction or maintenance of the articular cartilage phenotype. First generation, E1, E3 deleted, serotype 5 adenoviral vectors carrying the complete cDNAs for TGF-b1, bone morphogenetic protein (BMP) -2, BMP -4 , BMP- 7, Sox 9, indian hedgehog (IHH), and insulin-like growth factor-1 (IGF-1) were constructed using cre-lox recombination. As a source of MSCs, adherent, colony forming cells from the bone marrow of 3-week-old calves were isolated, cultured and used at low passage. Individual flasks of MSCs were infected with Ad.TGF-b1, Ad.BMP-2, Ad.BMP-4, Ad.BMP-7 and Ad.IGF-1 at doses sufficient to provide low ( 100 ng/ml) amounts of transgene expression per 24 hrs. Delivery of Sox-9 and Ad.IHH was performed over a wide range of viral doses. At 24 hours post infection, 2.0 x 10^5 cells of each culture were pelleted by centrifugation. The aggregates were cultured for 3 weeks in conical tubes in a defined, serum-free medium containing ascorbate 2-phosphate and dexamethasone. Chondrogenesis was determined using histologic staining for glycosaminoglycans and by immunohistochemistry for collagen types I and II. By this method, gene transfer and expression of BMP-2 and BMP-4 showed the greatest chondrogenic potential. This was followed to a lesser extent by TGF-b1 and then Sox-9 and IGF-1. Delivery of IHH or BMP-7 was met with only minimal chondrogenic activity which was only slightly greater than uninfected control cells. The potent activity of the secreted factors BMPs-2, -4 and TGF-b1 were expected and are consistent with the literature. The capacity to induce chondrogenesis through delivery of the Sox-9 transcription factor offers a promising intracellular alternative to the overexpression of secretable pleiotropic growth factors. The relative lack of chondroinduction following overexpression of IGF-1 and IHH is perhaps not surprising because these products thought to be more important for maintenance than induction of the chondrocytic phenotype. Future work will involve evaluation these genes in combinatorial studies.