The role of TGF-β signaling on differentiation of dental stem cells into endothelial cells and smooth muscle cells

Abstract

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 vascular smooth muscle cells) is a challenging task for tissue neovascularization. The transforming growth factor beta (TGF-β) signaling pathway plays an important role in the development of blood vessels, such as modulating the expression of vascular endothelial growth factor (VEGF) to regulate endothelial cells (ECs) differentiation and promoting vascular smooth muscle cells (vSMCs) differentiation through TGF-/Smad signaling pathway. Hence, we investigated whether stem cells from human exfoliated deciduous teeth (SHED) could be induced into functional ECs and vSMCs through regulating the TGF-β signaling pathway.

In our first study, we initially used VEGF-A to stimulate two dental pulp-derived stem cells (dental pulp stem cells [DPSCs] and SHED) and compared their differentiation capacity into ECs. We also investigated whether the TGF-β signaling inhibitor SB-431542 could enhance the inductive effect of VEGF-A on endothelial differentiation, as well as the underlying mechanisms involved. ECs differentiated from dental pulp-derived stem cells exhibited the typical phenotypes of primary ECs, with SHED possessing a higher endothelial differentiation potential than DPSCs. Compared with VEGF-A alone, the combination of VEGF-A and SB-431542 significantly enhanced the endothelial differentiation of SHED. The presence of SB-431542 inhibited the phosphorylation of Suppressor of Mothers Against Decapentaplegic 2/3 (SMAD2/3), allowing for VEGF-A-dependent phosphorylation and upregulation of VEGFR2. Our results indicate that the combination of VEGF-A and SB-431542 could enhance the differentiation of dental pulp-derived stem cells into endothelial cells, and this process is mediated through enhancement of VEGF-A-VEGFR2 signaling and concomitant inhibition of TGF-β-SMAD2/3 signaling.

In our second study, 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. By analyzing the expression of specific markers of SMCs, we confirmed that TGF-β1, and not BMP4, could induce SHED differentiation into SMCs. The differentiation efficiency was relatively high 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 partially related to the TGF-β1-ALK5 signaling pathway. In conclusion, the present findings demonstrated that the TGF-β signaling pathway might play an important role in regulating ECs and vSMCs differentiation from SHED. These findings thus provide a new insight for us into the usage of dental pulp-derived stem cells in vascular tissue engineering.