Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes

Saliya Gurusinghe, Nadeeka Bandara, Bryan Hilbert, Gareth Trope, Lexin Wang, Padraig Strappe

Research output: Contribution to journalArticle

Abstract

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.

LanguageEnglish
Pages9-19
Number of pages11
JournalGene
Volume680
DOIs
Publication statusPublished - 05 Jan 2019

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Chondrogenesis
Chondrocytes
Hypertrophy
Horses
Green Fluorescent Proteins
Messenger RNA
Transcription Factors
Fibrocartilage
Articular Cartilage
Mitosis
Transcriptional Activation
Cartilage
Extracellular Matrix
Intercellular Signaling Peptides and Proteins
Down-Regulation
Gene Expression

Cite this

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title = "Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes",
abstract = "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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Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes. / Gurusinghe, Saliya; Bandara, Nadeeka; Hilbert, Bryan; Trope, Gareth; Wang, Lexin; Strappe, Padraig.

In: Gene, Vol. 680, 05.01.2019, p. 9-19.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Lentiviral vector expression of Klf4 enhances chondrogenesis and reduces hypertrophy in equine chondrocytes

AU - Gurusinghe, Saliya

AU - Bandara, Nadeeka

AU - Hilbert, Bryan

AU - Trope, Gareth

AU - Wang, Lexin

AU - Strappe, Padraig

PY - 2019/1/5

Y1 - 2019/1/5

N2 - 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.

AB - 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.

KW - Chondrogenesis

KW - Hypertrophy

KW - Kruppel like factor 4 (Klf4)

KW - Lentiviral vector

KW - Transforming Growth Factor beta (TGFbeta)

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