Monolayer expansion of chondrocytes in culture results in the dedifferentiation of chondrocytes with inferior cartilage specific extracellular matrix synthesis and proliferation when compared with its native counterpart. We aimed to enhance chondrocyte proliferation and articular cartilage specific gene expression through ectopic expression of the major pluripotency transcription factors (Oct4, Sox2, Klf4 and c-Myc). We also aimed to provide insights to the modulation of TGFβ receptor mRNA with Klf4 overexpression. Equine chondrocytes pooled from three donors were transduced with lentiviral vectors expressing the induced pluripotency factors, Oct4, Sox2. Klf4 and c-Myc (OSKM), singly, or in combination or together with green fluorescent protein (GFP) as a control. Klf4 and c-Myc overexpressing chondrocytes showed a significant increase in mitosis when compared to the control (P < 0.01 and P < 0.0001 respectively). Furthermore, overexpression of Klf4 or OSKM in three dimensional (3D) culture of equine chondrocytes resulted in a significant increase in Col2a1 mRNA levels relative to the controls (P < 0.05 and P < 0.01 respectively) while all transcription factors significantly lowered the mRNA of the fibrocartilage marker Col1a1. We also employed a Col2a1 promoter driven GFP reporter for real time monitoring of Col2a1 gene activation in 3D micromass culture, which showed significantly higher promoter activity when cultures were treated with the growth factor TGFβ3 (P < 0.05). The chondrogenic properties of Klf4 transduced chondrocytes at a lower passage (P4) showed significant increases in Sox9 (P < 0.001), Col2a1 (P < 0.05) and TGFβ receptor I (P < 0.05) and II (P < 0.001) expression relative to the DS-Red expressing control. The chondrocyte dedifferentiation marker Col1a1 and hypertrophic marker Col10a1 were significantly downregulated with the inclusion of Klf4 (P < 0.01 and P < 0.05 respectively). In Conclusion, chondrogenic re-differentiation and proliferation of equine chondrocytes is promoted through ectopic expression of Klf4 while suppressing chondrocyte dedifferentiation.

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Nadeeka Bandara

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Department of Medicine

Dr

Nadeeka Bandara

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Nadeeka Bandara

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