Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors

© 2015, Springer Science+Business Media Dordrecht.Objectives: Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Results: Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). Conclusion: A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.