Type IIB Expression Research Articles

916. Genetically Modified Mesenchymal Stem Cells Expressing Transforming Growth Factor-β1 Develop a Cartilage Phenotype

INTRODUCTIONAcute cartilage injury and subsequent osteoarthritis are a debilitating and common syndrome in the aging population. Transplantation of MSCs to joints has been successfully used in small animal models. However, in extensive defects the application of MSCs improved defect filling with only fibrous tissue. Enhancing chondrogenesis of the MSCs by TGF-β1 gene transduction before transplantation may provide the impetus for better quality cartilaginous repair.MATERIALS AND METHODSAadenovirus vector encoding equine TGF-β1 (AdTGF-β1) was used to transfect MSCs. Monolayer cultures were transduced with AdTGF-β1, AdGFP, or no virus. Monolayers were pelleted after 24 hours, and 15 pellets harvested at 2, 7, 14, and 21 days. Chondrogenesis was determined by qPCR assessment of TGF-β1, aggrecan, coll IIA, and coll IIB gene expression. Medium was assayed for PG and TGF-β1. Confirmation of pellet chondrogenesis was made by HandE architecture, collagen II elaboration on immunohistochemistry, and toluidine blue PG staining.RESULTSPCR indicated AdTGFβ1 transfected MSCs expressed TGF-β1 RNA (Fig1Fig1), which induced markers of chondrogenic differentiationincluding aggrecan, and collagen types IIA and IIB (Figs 2Figs 2). Aggrecan expression was significantly enhanced on day 2. Immuno analysis detected collagen type II as early as 2 days after beginning aggregate cultures. This phenomenon continued through day 21, with intense collagen type II as the predominant collagen on day 21 (Fig 2Fig 2).Fig. 1View Large Image | View Hi-Res Image | Download PowerPoint SlideFig. 2View Large Image | View Hi-Res Image | Download PowerPoint SlideCollagen type I immuno analysis showed collagen type I deposition throughout all time periods, with increases under the influence of TGF-β1.DISCUSSIONSignificant increases in TGF-β1 expression were induced in AdTGF-β1 transduced MSC cells. Chondrogenic response was reflected in collagen type IIA and IIB expression which were significantly increased on day 7, 14 and 21. Longer term cultures showed a preponderance of collagen type IIB expression. Combined with morphologic evidence of strong chondrogenic condensation, these data provide molecular, histological and immunohistochemical evidence for substantial in vitro chondrogenic differentiation of equinebone-marrow derived MSCs. Based on these results AdTGF-β1 treated MSCs may be a robust source of chondrocytes for use in cartilage repair.

Effects of insulin-like growth factor-1 gene transfer on myosin heavy chains in denervated rat laryngeal muscle.

To determine whether the myotrophic activity of human insulin-like growth factor (hIGF)-1 promotes restoration of normal myosin heavy chain (MHC) composition after nerve injury, MHC composition was analyzed after hIGF-1 gene transfer in denervated rat laryngeal muscle. Animal model to study effects of gene transfer on laryngeal paralysis. In anesthetized rats, the left recurrent and superior laryngeal nerves are cut and suture ligated. A midline thyrotomy is performed, and the thyroarytenoid muscle is injected with a polyvinyl-based formulation containing a muscle specific expression system and hIGF-1 DNA (treatment group) or saline (control group). After 30 days, animals were killed, and the thyroarytenoid muscle was removed and processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric measurements were obtained to determine composition of MHCs. As previously described, MHC composition in denervated laryngeal muscle was characterized by a decrease in type IIB and IIL and up-regulation of IIA/IIX. Compared with controls, hIGF-1 treated animals demonstrated a significant increase in expression of type IIB and IIL and a significant decrease in expression of type IIA/X. These findings suggest that the myotrophic effect of hIGF-1 gene transfer results in normalization of MHC composition in denervated muscle, with suppression of type IIA/X MHC and promotion of type IIL expression.

Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide.

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.