Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, England, United Kingdom.
Matrix Biol. 2010 Oct;29(8):668-77. doi: 10.1016/j.matbio.2010.08.005. Epub 2010 Aug 22.
Tendon-like tissue generated from stem cells in vitro has the potential to replace tendons and ligaments lost through injury and disease. However, thus far, no information has been available on the mechanism of tendon formation in vitro and how to accelerate the process. We show here that human mesenchymal stem cells (MSCs) and bone marrow-derived mononuclear cells (BM-MNCs) can generate tendon-like tissue in 7days mediated by transforming growth factor (TGF) β3. MSCs cultured in fixed-length fibrin gels spontaneously synthesized narrow-diameter collagen fibrils and exhibited fibripositors (actin-rich, collagen fibril-containing plasma membrane protrusions) identical to those that occur in embryonic tendon. In contrast, BM-MNCs did not synthesize tendon-like tissue under these conditions. We performed real-time PCR analysis of MSCs and BM-MNCs. MSCs upregulated genes encoding type I collagen, TGFβ3, and Smad2 at the time of maximum contraction of the tendon-like tissue (7days). Western blot analysis showed phosphorylation of Smad2 at maximum contraction. The TGFβ inhibitor SB-431542, blocked the phosphorylation of Smad2 and stopped the formation of tendon-like tissue. Quantitative PCR showed that BM-MNCs expressed very low levels of TGFβ3 compared to MSCs. Therefore we added exogenous TGFβ3 protein to BM-MNCs in fibrin gels, which resulted in phosphorylation of Smad2, synthesis of collagen fibrils, the appearance of fibripositors at the plasma membrane, and the formation of tendon-like tissue. In conclusion, MSCs that self-generate TGFβ signaling or the addition of TGFβ3 protein to BM-MNCs in fixed-length fibrin gels spontaneously make embryonic tendon-like tissue in vitro within 7days.
由干细胞体外生成的肌腱样组织有可能替代因损伤和疾病而丧失的肌腱和韧带。然而，到目前为止，关于体外肌腱形成的机制以及如何加速这一过程的信息还没有。我们在这里表明，人骨髓间充质干细胞（MSCs）和骨髓源性单核细胞（BM-MNCs）可以通过转化生长因子（TGF）β3 在 7 天内生成肌腱样组织。在固定长度的纤维蛋白凝胶中培养的 MSC 会自发合成窄直径的胶原原纤维，并表现出与胚胎肌腱中出现的纤维突（富含肌动蛋白、含有胶原原纤维的质膜突起）相同的纤维突。相比之下，BM-MNCs 在这些条件下不会合成肌腱样组织。我们对 MSC 和 BM-MNCs 进行了实时 PCR 分析。MSC 在肌腱样组织最大收缩时（7 天）上调编码 I 型胶原、TGFβ3 和 Smad2 的基因。Western blot 分析显示 Smad2 在最大收缩时发生磷酸化。TGFβ 抑制剂 SB-431542 阻断 Smad2 的磷酸化并阻止肌腱样组织的形成。定量 PCR 显示，BM-MNCs 表达的 TGFβ3 水平远低于 MSC。因此，我们在纤维蛋白凝胶中向 BM-MNCs 添加外源性 TGFβ3 蛋白，导致 Smad2 磷酸化、胶原原纤维合成、质膜出现纤维突和肌腱样组织形成。总之，能够自我产生 TGFβ 信号的 MSC 或向固定长度纤维蛋白凝胶中的 BM-MNCs 添加 TGFβ3 蛋白，可在 7 天内自发地在体外生成胚胎样肌腱组织。
