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

Abstract

Background: 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).

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Angiogenesis; Dental pulp stem cells; Smooth muscle cells; Stemness; Tissue engineering.

Fig. 1

SMC-specific marker gene expression profile of SHED-derived SMCs induced with different concentrations and combinations of TGF-β1 and BMP4. Expression levels of SMC-specific marker genes relative to GAPDH with (a) different doses of TGF-β1 induction. There was significant upregulation of α-SMA and Calponin 1 gene expression levels as the TGF-β1 concentration is increased from 2.5 ng/ml to 10 ng/ml (p < 0.05). There were however no significant differences between the 10 ng/ml, 20 ng/ml and 30 ng/ml TGF-β1 treatment groups (p > 0.05). SM22α gene expression was a little different, by which 5 ng/ml TGF-β1 had a same effect with 10 ng/ml, 20 ng/ml and 30 ng/ml. b Different doses of BMP4 induction and with different combinations of TGF-β (10 ng/ml) and BMP4 (5, 10, 30 ng/ml). There were no significant differences amongst the different doses of BMP4 (p > 0.05), and also no significant differences amongst the different combinations of TGF-β and BMP4 (p > 0.05). c 10 ng/ml TGF-β1, 10 ng/ml BMP4 and the combination of 10 ng/ml TGF-β1 with BMP4. The effect of TGF-β1 was weakened when combined with BMP4 (p < 0.05). * ^ Φ: p < 0.05. B means BMP4; TB means TGF-β and BMP4. All experiments were performed three times (N = 3). BMP4 bone morphogenetic protein 4, TGF-β1 transforming growth factor beta 1, α-SMA alpha-smooth muscle actin

Fig. 2

Characterization of SHED-derived SMCs. a Expression levels of SMC-specific marker gene expression relative to GAPDH at different induction time points. The expression levels of all SMC-specific marker genes peaked on day 5 (p < 0.05, compared with other time points). At passage 2, the gene expression levels of α-SMA and Calponin 1 declined but were still significantly higher than the control group (p < 0.05); while the gene expression level of SM22α declined to the same level as the control group (p > 0.05). * ^ Φ: p < 0.05 compared to all other time points. b The protein expression levels of SM22α and Calponin 1 were analyzed by western blot utilizing β-actin as the internal marker. Numbers depict the band density normalized against untreated controls and β-actin. c SB-431542 suppressed TGF-β1-mediated SMC differentiation. * ^ Φ: p < 0.05 between TGF-β1 group and SB-431542 + TGF-β1-treated group. d Smad2/3 became phosphorylated within 30 minutes of exposure to TGF-β1, and SB-431542 inhibited this phosphorylation. Numbers depict the band density normalized against untreated controls and β-actin. e SHED-derived SMCs were also analyzed for expression of SMC-specific markers (α-SMA, Calponin, SM22α and SM-MHC) with flow cytometry (α-SMA+ 86.1%, SM22α + 93.9%, Calponin + 56.8%, and SM-MHC+ 88.2% respectively) (the isotype control in red) and (f) immunofluorescence staining (nuclei in blue). All experiments were performed three times (N = 3). SM-MHC smooth muscle-myosin heavy chain, SHED stem cells from human exfoliated deciduous teeth, α-SMA alpha-smooth muscle actin

Fig. 3

Collagen gel contraction assay. Gel surface areas were measured and further analyzed using the Image J software. *: p < 0.05, ^: p > 0.05. HUVECs human umbilical vein endothelial cells, SHED stem cells from human exfoliated deciduous teeth

Fig. 4

Vascular tube formation by SHED-derived SMCs and ECs on Matrigel. a Immunofluorescence image of vascular structure (EC in red; SHED-derived SMCs in green). (b) Phase-contrast images (×10) of vascular structures from 4 hours to 24 hours after seeding ECs and SHED-derived SMCs on Matrigel. HUVECs human umbilical vein endothelial cells, SHED stem cells from human exfoliated deciduous teeth, SMCs smooth muscle cells

Fig. 5

SHED-derived SMCs promote EC vessel formation. a Photomicrographs of EC-coated microbeads cultured in fibrin gels with SMC, SHED and SHED-induced SMC on the gel surface. b The schematic representation of calculating tube formation from each bead: (i) the number of vessels which sprout from the bead directly (in blue); (ii) the total number of individual vessel segments (including blue and yellow); and (iii) the total length of all the vessel segments. c Quantification of the vessel structures in the fibrin gel bead assay in the presence of SMC, SHED and SHED-induced SMC.*: p < 0.05 versus SMCs group, ^: p < 0.05 between SHED and SHED-derived SMCs. d Western blot showing fibronectin expression within each experimental group. All experiments were performed three times (N = 3). ECs endothelial cells, SHED stem cells from human exfoliated deciduous teeth, SMCs smooth muscle cells