Loebel Claudia, Czekanska Ewa M, Bruderer Marco, Salzmann Gian, Alini Mauro, Stoddart Martin J
1 AO Research Institute Davos , Davos Platz, Switzerland .
Tissue Eng Part A. 2015 Jan;21(1-2):115-23. doi: 10.1089/ten.TEA.2014.0096. Epub 2014 Aug 4.
Runx2 is one of the most studied transcription factors expressed in mesenchymal stem cells (MSCs) upon their commitment toward an osteogenic differentiation. During endochondral bone formation in vivo, Sox9 directly interacts with Runx2 and represses its activity; however, the role of Sox9 in direct osteogenesis in vitro has been largely overlooked.
Bone marrow-derived human MSCs (hMSCs) were cultured in vitro either in the control or osteogenic medium supplemented with dexamethasone (DEX). To further investigate the role of Sox9 in direct osteogenesis in vitro, hMSCs were treated with Sox9 siRNA.
We show here that Sox9 is the key early indicator during in vitro osteogenic differentiation of hMSCs. Osteogenic induction leads to a significant decrease of Sox9 gene and protein expression by day 7. Treatment of hMSCs with Sox9 siRNA enhanced mineralization in vitro, suggesting that downregulation of Sox9 is involved in direct osteogenesis. siRNA knockdown of Sox9 did not in itself induce osteogenesis in the absence of DEX, indicating that other factors are still required.
Screening of not preselected donors of different ages and gender (n=12) has shown that the Runx2/Sox9 ratio on day 7 is correlated to the (45)Ca incorporation on day 28. The impact of Sox9 downregulation in the mineralization of human MSCs in vitro indicates a so far unprecedented role of Sox9 as a major regulator of direct osteogenesis. We propose that the Runx2/Sox9 ratio is a promising, early, in vitro screening method for osteogenicity of human MSCs.
Runx2是在间充质干细胞（MSC）向成骨分化过程中研究最多的转录因子之一。在体内软骨内骨形成过程中，Sox9直接与Runx2相互作用并抑制其活性；然而，Sox9在体外直接成骨中的作用在很大程度上被忽视了。
将人骨髓来源的间充质干细胞（hMSC）在对照培养基或添加地塞米松（DEX）的成骨培养基中进行体外培养。为了进一步研究Sox9在体外直接成骨中的作用，用Sox9 siRNA处理hMSC。
我们在此表明，Sox9是hMSC体外成骨分化过程中的关键早期指标。成骨诱导导致第7天时Sox9基因和蛋白表达显著降低。用Sox9 siRNA处理hMSC可增强体外矿化，表明Sox9的下调参与直接成骨。在没有DEX的情况下，Sox9的siRNA敲低本身不会诱导成骨，这表明仍需要其他因素。
对不同年龄和性别的未预先选择的供体（n = 12）进行筛选表明，第7天时的Runx2/Sox9比值与第28天时的（45）Ca掺入相关。Sox9下调对人MSC体外矿化的影响表明，Sox9作为直接成骨的主要调节因子具有迄今为止前所未有的作用。我们提出，Runx2/Sox9比值是一种有前景的、早期的体外筛选人MSC成骨能力的方法。
