Department of Bone and Cartilage Biology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Biochem Biophys Res Commun. 2011 Aug 5;411(3):607-12. doi: 10.1016/j.bbrc.2011.06.194. Epub 2011 Jul 6.
There is a significant need for cell sources for cartilage regenerative medicine. It has been reported that the combined transduction of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) directly induces chondrogenic cells from mouse dermal fibroblast (MDF) culture. To gain insights into the process by which cellular characteristics are altered by transduction of c-Myc, Klf4 and SOX9, we examined marker gene expression in the MDF culture at various time points after transduction. The expression of fibroblast-markers was reduced first, followed by an increase in the expression of a chondrocyte-marker. We detected no expression of pluripotent markers at any time point examined. To determine whether or not induced chondrogenic cells go through a pluripotent state after transduction, we analyzed MDFs prepared from Nanog-GFP transgenic mice by monitoring expression of the GFP-labeled pluripotent marker Nanog-GFP in the MDF culture, using time-lapse microscopic observation. Whole-well time-lapse observation revealed that none of the induced chondrogenic cells displayed GFP fluorescence during induction. These results indicate that cells do not undergo a pluripotent state during direct induction of chondrogenic cells from fibroblast culture by transduction of c-Myc, Klf4 and SOX9.
对于软骨再生医学来说，细胞来源是一个重大的需求。据报道，两种重编程因子（c-Myc 和 Klf4）和一种软骨生成因子（SOX9）的联合转导可直接诱导来自小鼠真皮成纤维细胞（MDF）培养物的软骨细胞。为了深入了解 c-Myc、Klf4 和 SOX9 转导改变细胞特征的过程，我们在转导后不同时间点检查了 MDF 培养物中的标记基因表达。首先减少成纤维细胞标志物的表达，然后增加软骨细胞标志物的表达。在任何检查的时间点都未检测到多能标志物的表达。为了确定诱导的软骨细胞在转导后是否经过多能状态，我们通过监测 GFP 标记的多能标志物 Nanog-GFP 在 MDF 培养物中的表达，使用延时显微镜观察，分析来自 Nanog-GFP 转基因小鼠的 MDF。全孔延时观察显示，在诱导过程中，没有一个诱导的软骨细胞显示 GFP 荧光。这些结果表明，在 c-Myc、Klf4 和 SOX9 的联合转导下，直接从成纤维细胞培养物诱导软骨细胞时，细胞不会经历多能状态。
