Department of Orthopedics and Rehabilitation and Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
PLoS One. 2011;6(7):e22887. doi: 10.1371/journal.pone.0022887. Epub 2011 Jul 27.
Human mesenchymal stem cells (hMSCs) are multipotent cells capable of differentiating into a variety of mature cell types, including osteoblasts, adipocytes and chondrocytes. It has previously been shown that, when expanded in medium supplemented with fibroblast growth factor-2 (FGF-2), hMSCs show enhanced chondrogenesis (CG). Previous work concluded that the enhancement of CG could be attributed to the selection of a cell subpopulation with inherent chondrogenic potential. In this study, we show that FGF-2 pretreatment actually primed hMSCs to undergo enhanced CG by increasing basal Sox9 protein levels. Our results show that Sox9 protein levels were elevated within 30 minutes of exposure to FGF-2 and progressively increased with longer exposures. Further, we show using flow cytometry that FGF-2 increased Sox9 protein levels per cell in proliferating and non-proliferating hMSCs, strongly suggesting that FGF-2 primes hMSCs for subsequent CG by regulating Sox9. Indeed, when hMSCs were exposed to FGF-2 for 2 hours and subsequently differentiated into the chondrogenic lineage using pellet culture, phosphorylated-Sox9 (pSox9) protein levels became elevated and ultimately resulted in an enhancement of CG. However, small interfering RNA (siRNA)-mediated knockdown of Sox9 during hMSC expansion was unable to negate the prochondrogenic effects of FGF-2, suggesting that the FGF-2-mediated enhancement of hMSC CG is only partly regulated through Sox9. Our findings provide new insights into the mechanism by which FGF-2 regulates predifferentiation hMSCs to undergo enhanced CG.
人骨髓间充质干细胞（hMSCs）是多能细胞，能够分化为多种成熟细胞类型，包括成骨细胞、脂肪细胞和成软骨细胞。先前的研究表明，当在含有成纤维细胞生长因子-2（FGF-2）的培养基中扩增时，hMSCs 表现出增强的软骨形成（CG）。先前的工作得出结论，CG 的增强可能归因于具有固有软骨形成潜力的细胞亚群的选择。在这项研究中，我们表明 FGF-2 预处理实际上通过增加 Sox9 蛋白的基础水平来启动 hMSCs 进行增强的 CG。我们的结果表明，暴露于 FGF-2 后 30 分钟内 Sox9 蛋白水平升高，并随着暴露时间的延长而逐渐增加。此外，我们通过流式细胞术表明，FGF-2 增加了增殖和非增殖 hMSCs 中 Sox9 蛋白的水平，这强烈表明 FGF-2 通过调节 Sox9 来启动 hMSCs 随后的 CG。事实上，当 hMSCs 暴露于 FGF-2 2 小时后，通过球状体培养将其分化为软骨谱系时，磷酸化 Sox9（pSox9）蛋白水平升高，最终导致 CG 增强。然而，在 hMSC 扩增过程中使用小干扰 RNA（siRNA）介导的 Sox9 敲低无法否定 FGF-2 的前成软骨作用，这表明 FGF-2 介导的 hMSC CG 增强仅部分通过 Sox9 调节。我们的发现为 FGF-2 调节预分化 hMSCs 进行增强的 CG 的机制提供了新的见解。
