The in vitro expansion of chondrocytes results in cellular dedifferentiation with reduced expression of the chondrogenic differentiation factor Sox9 and extracellular matrix proteins. Viral based transduction of chondrocytes with Sox9, along with exposure to Transforming Growth Factor Beta (TGFβ) has been shown to enhance the chondrogenic phenotype in culture expanded chondrocytes, preventing dedifferentiation. Recent studies have demonstrated that expression of stem cell factors Klf-4 and c-Myc together with Sox9 bypasses the pluripotent state and differentiates fibroblasts directly into chondrocytes. In this study we demonstrate that lentiviral vector expression of Sox9 either alone or together with induced pluripotent stem cell transcription factors (Oct4, c-Myc, Sox2 and Klf4) maintains the chondrocyte phenotype in culture expanded chondrocytes when used in conjunction with the growth factor TGFβ-3.
Culture expanded chondrocytes were transduced with lentiviral vectors expressing Sox9 or with a lentiviral vector co-expressing the 4 induced pluripotent stem (iPS) cell factors , Oct4, Sox2, Klf4 and c-Myc. High density pellet cultures were formed with transduced cells and incubated in chondrogenic media supplemented with TGFβ-3 for 7 days. Chondrogenic capacity was evaluated by reverse transcriptase polymerase chain reaction (RT-PCR) of candidate genes, immunohistochemistry and histology.
Sox9 and iPS stem cell factor transduced cultures resulted in an increase in pellet size compared to controls and revealed extracellular matrix accumulation with intense staining with toluidine blue and safranin O. A similar trend was observed with quantitative assessment for glycosaminoglycan. RT-PCR was carried out for col2a1 and the hypertrophic marker col10a1, with an increase in col2aI signal observed in Sox9 transduced cultures. In contrast the expression of col10a1 was lower in the gene modified cells.
Lentiviral vector transduction of articular chondrocytes with iPS stem cell factors and Sox9 enables stable culture expansion of chondrocytes and enhances the chondrocyte phenotype with the supplementation of TGFβ-3.