TGF-β1-induced differentiation of SHED into functional smooth muscle cells

Jian Guang Xu, Shao Yue Zhu, Boon Chin Heng, Waruna Lakmal Dissanayaka, Cheng Fei Zhang

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.