Xu Jian Guang, Zhu Shao Yue, Heng Boon Chin, Dissanayaka Waruna Lakmal, Zhang Cheng Fei
Comprehensive Dental Care, Endodontics, Faculty of Dentistry, The University of Hong Kong, Pokfulam, Hong Kong, China.
HKU Shenzhen Institute of Research and Innovation, Hong Kong, China.
Stem Cell Res Ther. 2017 Jan 23;8(1):10. doi: 10.1186/s13287-016-0459-0.
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.
充足的血管化对于新鲜移植的组织工程构建体供应营养和排出代谢废物至关重要。获取血管组织工程的合适构建单元（即内皮细胞和平滑肌细胞）是组织新生血管形成的一项具有挑战性的任务。因此，我们研究了人脱落乳牙干细胞（SHED）是否可被诱导分化为功能性血管平滑肌细胞（vSMC）。
我们利用转化生长因子β家族的两种细胞因子，即转化生长因子β1（TGF-β1）和骨形态发生蛋白4（BMP4），诱导SHED分化为平滑肌细胞。采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、蛋白质印迹法和免疫染色分析蛋白质表达。此外，为了检查这些源自SHED的平滑肌细胞是否具有与原代平滑肌细胞相同的功能，在此使用了体外基质胶血管生成试验、纤维蛋白凝胶珠试验和功能收缩研究。
通过分析平滑肌细胞特异性标志物（α-SMA、SM22α、钙调蛋白和SM-MHC）的表达，我们证实TGF-β1而非BMP4可诱导SHED分化为平滑肌细胞。通过流式细胞术评估，分化效率相对较高（α-SMA为86.1%，SM22α为93.9%，钙调蛋白为56.8%，SM-MHC为88.2%）。体外基质胶血管生成试验表明，源自SHED的平滑肌细胞和人脐静脉内皮细胞（HUVEC）生成的血管结构在血管稳定性方面与原代平滑肌细胞和HUVEC相当。纤维蛋白凝胶珠试验表明，与原代平滑肌细胞相比，源自SHED的平滑肌细胞具有更强的促进血管形成的能力。对纤维蛋白凝胶中蛋白质表达的进一步分析表明，含有源自SHED的平滑肌细胞的培养物中纤连蛋白的表达水平高于原代平滑肌细胞组。此外，还证实源自SHED的平滑肌细胞表现出功能性收缩。当给予ALK5的特异性抑制剂SB-431542时，TGF-β1刺激不能诱导SHED分化为平滑肌细胞，表明SHED向平滑肌细胞的分化在某种程度上与TGF-β1-ALK5信号通路有关。
SHED可成功诱导分化为用于血管组织工程的功能性平滑肌细胞，且这一过程可通过ALK5信号通路进行调控。因此，SHED似乎是血管组织工程中一种有前景的候选细胞类型。
