Department of Cardiothoracic Surgery, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
FASEB J. 2019 Apr;33(4):5641-5653. doi: 10.1096/fj.201802137RRR. Epub 2019 Jan 29.
Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long-segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF-β3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF-β3 induction. hUCMSCs were preconditioned with 1 μM of KGN for 3 d, sequentially pelleted, and incubated with TGF-β3 for 28 d. Then, the expression of chondrogenesis- and ossification-related genes was evaluated by immunohistochemistry and RT-PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and β-catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up-regulated in hUCMSCs. Furthermore, the KGN-preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene ( SOX9, aggrecan, and collagen II) expression and reduced expression of ossific genes (collagen X and MMP13) compared with hUCMSCs treated with TGF-β3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of β-catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt-related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the β-catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN-preconditioned hUCMSCs and TGF-β3 encapsulated electrospun poly( l-lactic acid-co-ε-caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed β-catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF-β3 induction might be preferable for cartilage engineering strategies using MSCs.-Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X. Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and β-catenin-related pathways.
软骨工程策略使用间充质干细胞（MSCs）可以提供更好的解决方案来解决长段气管缺损问题。然而，广泛使用的含 TGF-β3 的软骨形成方案存在效率低下和细胞表型不稳定等缺点，这是一个问题。在我们的研究中，为了优化人脐带间充质干细胞（hUCMSCs）的软骨分化，在 TGF-β3 诱导前进行了卡托金（KGN）预处理。hUCMSCs 用 1 μM 的 KGN 预处理 3 天，然后进行沉淀，并用 TGF-β3 孵育 28 天。然后，通过免疫组织化学和 RT-PCR 评估软骨形成和骨化相关基因的表达。通过磷酸化激酶筛选探索 KGN 预处理有益效果的潜在机制，并使用 JNK 抑制剂（SP600125）和 β-连环蛋白激活剂（SKL2001）在体外和体内进行验证。KGN 预处理后，纤维母细胞生长因子受体 3（一种前软骨干细胞标志物）在 hUCMSCs 中的表达上调。此外，与仅用 TGF-β3 处理的 hUCMSCs 相比，KGN 预处理的 hUCMSCs 能有效地分化为软骨细胞，具有更高的软骨形成基因（SOX9、聚集蛋白和 II 型胶原）表达和降低的成骨基因（X 型胶原和 MMP13）表达。磷酸激酶筛选表明，KGN 预处理的有益效果与 JNK 磷酸化的上调和 β-连环蛋白水平的抑制直接相关。阻断和激活试验表明，KGN 预处理的促软骨形成作用主要通过激活 JNK/Runt 相关转录因子（RUNX）1 通路来实现，而抗成骨作用则通过抑制 β-连环蛋白/RUNX2 通路来实现。最终，基于 KGN 预处理的 hUCMSCs 和包埋在电纺聚（L-乳酸-co-ε-己内酯）/胶原纳米纤维中的 TGF-β3 的气管贴片成功用于修复兔模型中的气管缺损。总之，KGN 预处理可能通过促进 hUCMSCs 向软骨前阶段分化，增强 JNK 磷酸化和抑制 β-连环蛋白，从而提高 hUCMSCs 的软骨分化能力。该方案由 KGN 预处理和随后的 TGF-β3 诱导组成，可能更适合于使用 MSCs 的软骨工程策略。
