Invitro culture expansion of chondrocytes is essential for generating sufficient cell numbers for transplantation. However, extended culture can lead to loss of the articular phenotype, with reduced expression of Col2A1. The pluripotency transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) have been shown to reprogram fibroblasts into chondrocytes. In this study we show that lentiviral vector expression of OSKM in dedifferentiated equine chondrocytes enhances the articular chondrocyte phenotype in 3D cultures resulting in high levels of extracellular matrix synthesis.Monolayer expanded chondrocytes were transduced with a lentiviral vector co-expressing Oct4, Sox2, Klf4 and c-Myc (OSKM) or separately with a lentiviral vector expressing Green fluorescence protein (GFP). Transduced chondrocytes were induced in 3D pellet cultures for 14 days with media containing transforming growth factor beta 3 (TGFβ-3). Glycosaminoglycan (GAG) was quantified using Dimethyl methylene blue (DMMB). Immunohistochemical staining was performed on paraffin embedded sections for collagen type II and collagen type X and QPCR was also performed to quantify transcripts of the same targets. Toluidine blue staining for GAG showed intense staining in chondrocytes expressing OSKM. The total DNA and GAG content was significantly higher in the OSKM transduced cells compared to untransduced chondrocytes (p<0.05). Immunohistochemical staining for collagen type II showed diffused extracellular matrix in OSKM transduced cells compared to the chondrocytes controls with Collagen X expression evident in all conditions. mRNA expression levels of col2a1 and col10a1 were significantly higher in OSKM expressing cells indicating redifferentiation of the chondrocytes with ectopic expression of the transcription factors (p<0.0001).
|Publication status||Published - 01 Sep 2015|
|Event||4th TERMIS World Congress - Boston Marriott Copley Place, Boston, United States|
Duration: 08 Sep 2015 → 11 Sep 2015
|Conference||4th TERMIS World Congress|
|Abbreviated title||PAST, PRESENT, FUTURE: The Evolution of Regenerative Medicine|
|Period||08/09/15 → 11/09/15|