The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.
The Effects of the WNT-Signaling Modulators BIO and PKF118-310 on the Chondrogenic Differentiation of Human Mesenchymal Stem Cells.
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DOI：
10.3390/ijms19020561
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发表时间：
2018-02-13
影响因子：
5.6
通讯作者：
Karperien M
中科院分区：
文献类型：
--
作者：
Huang X;Zhong L;Hendriks J;Post JN;Karperien M
骨髓间充质干细胞（Mesenchymal stem cells，MSCs）是一种多能干细胞，主要来源于骨髓，是骨和软骨组织工程的理想细胞来源。MSC的成软骨分化的研究对于基于MSC的软骨再生特别感兴趣。在本研究中，我们的目的是通过使用小分子WNT抑制剂PKF 118 -310和激活剂BIO调节WNT信号来优化MSC的时间诱导分化的条件。从骨髓抽吸物中分离人间充质干细胞（hMSCs），并在hMSCs增殖培养基中培养。随后建立颗粒培养物用于5周的三维成软骨分化。小分子糖原合成酶激酶-3抑制剂6-溴靛玉红-3-肟（BIO）可增加WNT信号传导，WNT抑制剂PKF 118 -310（PKF）可降低WNT信号传导。通过实时荧光定量PCR、组织学方法和ELISA检测BIO和PKF对hMSCs软骨形成的影响。我们发现，经典的WNT信号的激活BIO显着下调软骨特异性基因SOX 9，COL 2A 1和ACAN，和基质金属蛋白酶基因MMP 1/3/9/13的表达，但增加ADAMTS 4/5。PKF抑制WNT信号传导增加了SOX 9、COL 2A 1、ACAN和MMP 9的表达，但降低了MMP 13和ADAMTS 4/5的表达。此外，高水平的WNT信号诱导肥大标志物COL 10A 1、ALPL和RUNX 2、去分化标志物COL 1A 1以及糖酵解基因GULT 1和PGK 1的表达。沉淀颗粒基质中的糖胺聚糖（GAG）和II型胶原蛋白的沉积在BIO治疗组中显著丢失，在PKF治疗组中增加。在BIO组中，COL 10A 1的蛋白水平也被高度诱导。有趣的是，在软骨形成过程中，BIO减少了凋亡细胞的数量，而PKF显著诱导了凋亡。PKF处理后，细胞培养上清中天然WNT拮抗剂DKK 1和MMP 1蛋白水平下降。所有这些软骨形成作用似乎都是通过经典的WNT信号通路介导的，因为靶基因Axin 2和其他WNT成员，如TCF 4和β-连环蛋白，分别被BIO上调和PKF下调，BIO诱导β-连环蛋白的核转位，而PKF抑制β-连环蛋白转位到细胞核中。我们的结论是，加入BIO的hMSCs的软骨形成培养基导致软骨形成的损失，而PKF诱导软骨分化和软骨基质沉积，并抑制肥大分化。BIO通过抑制细胞凋亡促进细胞存活，而PKF则诱导细胞凋亡。该结果表明，WNT信号的过表达或过度抑制在一定程度上对软骨形成分化造成有害影响。在hMSC软骨形成过程中，WNT信号的临界水平的调节可能有利于软骨组织工程。
Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.
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