Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.
St. Vincent's Institute for Medical Research, Melbourne, VIC 3000, Australia.
Gene. 2019 Jan 5;680:9-19. doi: 10.1016/j.gene.2018.09.013. Epub 2018 Sep 8.
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.
软骨细胞在培养中的单层扩增会导致软骨细胞去分化，与天然软骨细胞相比，其合成和增殖的软骨特异性细胞外基质质量下降。我们的目的是通过异位表达主要多能转录因子（Oct4、Sox2、Klf4 和 c-Myc）来增强软骨细胞的增殖和关节软骨特异性基因表达。我们还旨在通过 Klf4 过表达来深入了解 TGFβ 受体 mRNA 的调节。从三个供体中汇集的马软骨细胞被慢病毒载体转导，该载体表达诱导多能性因子 Oct4、Sox2、Klf4 和 c-Myc（OSKM），单独或组合或与绿色荧光蛋白（GFP）一起作为对照。与对照组相比，Klf4 和 c-Myc 过表达的软骨细胞有丝分裂明显增加（分别为 P<0.01 和 P<0.0001）。此外，在马软骨细胞的三维（3D）培养中过表达 Klf4 或 OSKM 导致 Col2a1 mRNA 水平相对于对照组显著增加（分别为 P<0.05 和 P<0.01），而所有转录因子均显著降低了纤维软骨标志物 Col1a1 的 mRNA。我们还使用 Col2a1 启动子驱动的 GFP 报告基因实时监测 3D 微团培养中的 Col2a1 基因激活，结果表明，当用生长因子 TGFβ3 处理培养物时，启动子活性显著增加（P<0.05）。在较低传代（P4）时转导 Klf4 的软骨细胞的软骨生成特性显示 Sox9（P<0.001）、Col2a1（P<0.05）和 TGFβ 受体 I（P<0.05）和 II（P<0.001）表达显著增加与表达 DS-Red 的对照物相比。软骨细胞去分化标志物 Col1a1 和肥大标志物 Col10a1 的表达明显下调，加入 Klf4 后（分别为 P<0.01 和 P<0.05）。总之，通过异位表达 Klf4 促进了马软骨细胞的软骨再分化和增殖，同时抑制了软骨细胞去分化。
