TGF-β1-induced differentiation of SHED into functional smooth muscle cells.
TGF-β1-induced differentiation of SHED into functional smooth muscle cells.
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TGF-β1 诱导 SHED 分化为功能性平滑肌细胞。
DOI：
10.1186/s13287-016-0459-0
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发表时间：
2017-01-23
影响因子：
7.5
通讯作者：
Zhang CF
中科院分区：
文献类型：
--
作者：
Xu JG;Zhu SY;Heng BC;Dissanayaka WL;Zhang CF
组织工程支架的血管化是其营养供应和代谢废物排出的关键。获得适当的血管组织工程（即内皮细胞和壁细胞）的积木是一个具有挑战性的任务组织新生血管。因此，我们研究了来自人类脱落乳牙（SHED）的干细胞是否可以被诱导分化为功能性血管平滑肌细胞（vSMCs）。我们利用TGF-β家族的两种细胞因子，转化生长因子β 1（TGF-β1）和骨形态发生蛋白4（BMP 4），诱导SHED分化为SMC。使用定量实时聚合酶链反应（RT-qPCR）评估mRNA表达，并使用流式细胞术、蛋白质印迹和免疫染色分析蛋白质表达。此外，为了检查这些SHED衍生的SMCs是否具有与原代SMCs相同的功能，本文使用体外Matrigel血管生成测定、纤维蛋白凝胶珠测定和功能收缩研究。通过分析SMC特异性标志物（α-SMA、SM 22 α、Calponin和SM-MHC）的表达，我们证实TGF-β1而非BMP 4可以诱导SHED向SMC分化。流式细胞仪检测结果显示，α-SMA+86.1%，SM 22 α+93.9%，Calponin+56.8%，SM-MHC+ 88.2%的分化效率较高。体外Matrigel血管生成实验显示，SHED来源的SMC和人脐静脉内皮细胞（HUVECs）生成的血管结构在血管稳定性方面与原代SMC和HUVECs相当。纤维素凝胶微球法显示，与原代平滑肌细胞相比，SHED来源的平滑肌细胞具有更强的促血管形成能力。纤维蛋白凝胶中蛋白质表达的进一步分析表明，含有SHED衍生的SMC的培养物比原代SMC组表现出更高的纤连蛋白表达水平。此外，还证实了SHED衍生的SMC表现出功能性收缩性。当给予ALK 5特异性抑制剂SB-431542时，TGF-β1刺激不能诱导SHED向SMC分化，表明SHED向SMC的分化在某种程度上与TGF-β1-ALK 5信号通路有关。SHED可成功诱导为具有血管组织工程功能的平滑肌细胞，其诱导过程可通过ALK 5信号通路进行调控。因此，SHED似乎是一种有前途的候选细胞类型的血管组织工程。本文的在线版本（doi：10.1186/s13287-016-0459-0）包含补充材料，可供授权用户使用。
Adequate vascularization is crucial for supplying nutrition and discharging metabolic waste in freshly transplanted tissue-engineered constructs. Obtaining the appropriate building blocks for vascular tissue engineering (i.e. endothelial and mural cells) is a challenging task for tissue neovascularization. Hence, we investigated whether stem cells from human exfoliated deciduous teeth (SHED) could be induced to differentiate into functional vascular smooth muscle cells (vSMCs). We utilized two cytokines of the TGF-β family, transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein 4 (BMP4), to induce SHED differentiation into SMCs. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to assess mRNA expression, and protein expression was analyzed using flow cytometry, western blot and immunostaining. Additionally, to examine whether these SHED-derived SMCs possess the same function as primary SMCs, in vitro Matrigel angiogenesis assay, fibrin gel bead assay, and functional contraction study were used here. By analyzing the expression of specific markers of SMCs (α-SMA, SM22α, Calponin, and SM-MHC), we confirmed that TGF-β1, and not BMP4, could induce SHED differentiation into SMCs. The differentiation efficiency was relatively high (α-SMA+ 86.1%, SM22α+ 93.9%, Calponin+ 56.8%, and SM-MHC+ 88.2%) as assessed by flow cytometry. In vitro Matrigel angiogenesis assay showed that the vascular structures generated by SHED-derived SMCs and human umbilical vein endothelial cells (HUVECs) were comparable to primary SMCs and HUVECs in terms of vessel stability. Fibrin gel bead assay showed that SHED-derived SMCs had a stronger capacity for promoting vessel formation compared with primary SMCs. Further analyses of protein expression in fibrin gel showed that cultures containing SHED-derived SMCs exhibited higher expression levels of Fibronectin than the primary SMCs group. Additionally, it was also confirmed that SHED-derived SMCs exhibited functional contractility. When SB-431542, a specific inhibitor of ALK5 was administered, TGF-β1 stimulation could not induce SHED into SMCs, indicating that the differentiation of SHED into SMCs is somehow related to the TGF-β1-ALK5 signaling pathway. SHED could be successfully induced into functional SMCs for vascular tissue engineering, and this course could be regulated through the ALK5 signaling pathway. Hence, SHED appear to be a promising candidate cell type for vascular tissue engineering. The online version of this article (doi:10.1186/s13287-016-0459-0) contains supplementary material, which is available to authorized users.