title: Generation of Immortalized Equine Chondrocytes With Inducible Sox9 Expression Allows Control of Hypertrophic Differentiation.
abstract: Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower expression of chondrocyte specific collagen II mRNA (P < 0.0001) and up regulation of the hypertrophic marker collagen X (P < 0.0001) in three dimensional cultures. A Col2a1 promoter driven GFP reporter was constructed for real time monitoring of chondrogenic differentiation and a significant increase in promoter activation was observed in cultures treated with the growth factor TGFβ-3 (P < 0.05). To recapitulate the native articular chondrocyte phenotype we further transduced large T antigen immortalized chondrocytes with lentiviral vectors allowing either constitutive or doxycycline inducible expression of Sox9. In 3D cultures, the Sox9 over-expressing chondrocytes secreted significantly higher levels of extracellular matrix polysaccharide glycosaminoglycan (P < 0.05), while up-regulating collagen II and Aggrecan mRNA (P < 0.05) in both expression systems with a similar patterns observed with imunohistochemical staining. High levels of collagen X mRNA and protein were maintained with constitutive sox9 reflecting hypetrophic differentiation but significantly lower expression could be achieved with inducible Sox9. In conclusion, immortalization of equine chondrocytes results in stable proliferation but a reduction of chondrogenic potential whilst modulation of sox9 expression enabled control of hypertrophic characteristics. J. Cell. Biochem. 118: 1201-1215, 2017. © 2016 Wiley Periodicals, Inc.
authors: School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.|School of Animal and Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.
authors: Saliya
authors: S
authors: Gurusinghe
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.
authors: Bryan
authors: B
authors: Hilbert
authors: School of Animal and Veterinary Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.
authors: Gareth
authors: G
authors: Trope
authors: School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.
authors: Lexin
authors: L
authors: Wang
authors: School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.|O'Brien Institute Department, St. Vincent's Institute of Medical Research, Victoria 3065, Fitzroy, Australia.
authors: Nadeeka
authors: N
authors: Bandara
authors: School of Biomedical Sciences, Charles Sturt University, Locked Bag 588, Wagga Wagga, New South Wales 2650, Australia.
authors: Padraig
authors: P
authors: Strappe
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line / Q000166:cytology* / Q000378:metabolism; D049109:Cell Proliferation; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics; D024121:Collagen Type X / Q000235:genetics; D006025:Glycosaminoglycans / Q000378:metabolism; D006736:Horses; D055758:SOX9 Transcription Factor / Q000378:metabolism*
chemical_list: D024043:Collagen Type II; D024121:Collagen Type X; D006025:Glycosaminoglycans; D055758:SOX9 Transcription Factor
keywords:  LENTIVIRAL VECTOR; CHONDROCYTE; HYPERTROPHY; SOX9; T ANTIGEN; TELOMERASE REVERSE TRANSCRIPTASE