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

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Abstract

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.

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Acknowledgements

This work was supported by Charles Sturt University Faculty of Science (COMPACT) Post-Graduate Scholarship Award.

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Authors and Affiliations

  1. School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia

    Saliya Gurusinghe & Padraig Strappe

  2. School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia

    Peter Young & Jacob Michelsen

  3. Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW, Australia

    Padraig Strappe

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  1. Saliya Gurusinghe
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  2. Peter Young
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  3. Jacob Michelsen
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  4. Padraig Strappe
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Correspondence to Padraig Strappe.

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Gurusinghe, S., Young, P., Michelsen, J. et al. Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors. Biotechnol Lett 37, 1495–1504 (2015). https://doi.org/10.1007/s10529-015-1805-5

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  • DOI: https://doi.org/10.1007/s10529-015-1805-5

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