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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (5): 900-906. doi: 10.19723/j.issn.1671-167X.2019.05.018

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Comparative study of differentiation potential of mesenchymal stem cells derived from orofacial system into vascular endothelial cells

Jing XIE1,Yu-ming ZHAO1,Nan-quan RAO1,Xiao-tong WANG2,Teng-jiao-zi FANG1,Xiao-xia LI1,Yue ZHAI1,Jing-zhi LI1,Li-hong GE1,Yuan-yuan WANG1,△()   

  1. 1. Department of Pediatric Dentistry, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
    2. Department of Oral Emergency, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2017-10-10 Online:2019-10-18 Published:2019-10-24
  • Contact: Yuan-yuan WANG E-mail:cwyyd@126.com
  • Supported by:
    Supported by National Natural Science Youth Foundation of China(81500837)

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Abstract:

Objective: To compare the proliferation and capacity of differentiation to vascular endothelial cells and angiogenesis induction among stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC) and human bone marrow mesenchymal stem cells (BMSC) from orofacial bone. Methods: SHED and DPSC were isolated from pulp tissue of the patients. BMSC were isolated from orthognathic or alveolar surgical sites. The surface markers of the cells were detected by flowcytometry. Cell counting kit-8 (CCK-8) assays were conducted to detect the proliferation ability of the cells. The cells were induced into endothelial cells with conditional medium and then the induced cells were cultured in Matrigel medium. The expression of angiogenesis-related genes such as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2) and von Willebrand Factor (vWF) were quantified by real-time PCR. The cells were cultured in chick embryo chorioallantoic membrane (CAM) and the vessels were counted after 5 days. Results: The cell surface markers CD73, CD90, CD105 and CD146 of all the stem cells were positive, CD34 and CD45 were negative. The CD146 positive rate of SHED and DPSC was higher than that of BMSC. SHED had a higher proliferation rate than DPSC and BMSC. After angiogenic induction for 14 d, 3 kinds of cells emanated pseudopodia formed grid structure long vasculature in Matrigel media. The total length of tube formation of induced BMSC (7 759.7 μm) and SHED (7 734.3 μm) was higher than DPSC (5 541.0 μm). The meshes number of induced SHED (70.7) was higher than DPSC (60) and BMSC (53.7) in Matrigel medium. The expression of CD31, VEGFR2 and vWF genes of SHED were higher than those of BMSC and DPSC. VEGFR1 gene expression of BMSC was higher than that of the other groups, and SHED was higher than DPSC. The expression of VEGF showed no difference among the cells. No deference was showed between the effect of the stem cells and negative control on new formed vessels in CAM. The total length of vessels of SHED (30.4 mm) was higher than that of the negative control (20.9 mm) and BMSC (28.0 mm). Conclusion: SHED, DPSC and BMSC can differentiate into vascular endothelial cells. SHED showed a stronger angiogenesis differentiation and proliferation potential compared with DPSC and BMSC.

Key words: Stem cells from human exfoliated deciduous teeth, Dental pulp stem cells, Bone marrow mesenchymal stem cells, Differentiation into vascular endothelial cells, Cell proliferation

CLC Number: 

  • R78

Table 1

Specific primer for real-time PCR"

Gene Primer(5' to 3') Size/bp
CD31 F: TGTCAAGTAAGGTGGTGGAGTCT
R: AGGCGTGGTTGGCTCTGTT		 222
VEGF F: CCCACTGAGGAGTCCAACAT
R: AAATGCTTTCTCCGCTCTGA		 296
VEGFR1 F: ACCTCACTGCCACTCTAATTGTCA
R: AAGTCACACCTTGCTTCGGAATG		 196
VEGFR2 F: ACGGACAGTGGTATGGTTCTTGCC
R: GGTAGCCGCTTGTCTGGTTTGAG		 145
vWF F: GGGGTCATCTCTGGATTCAAG
R: TCTGTCCTCCTCTTAGCTGAA		 145
GAPDH F: CTGGGCTACACTGAGCACC
R: AAGTGGTCGTTGAGGGCAATG		 101

Figure 1

Surface marker of BMSC, DPSC and SHED observed by flow cytometry The cell surface markers CD73, CD90, CD105 and CD146 of all stem cells were positive, CD34 and CD45 were negative. SHED, stem cells from human exfoliated deciduous teeth; DPSC, dental pulp stem cell; BMSC, bone marrow mesenchymal stem cell."

Figure 2

The growth curve of BMSC, DPSC and SHED during a 9-day period CCK-8 assay, SHED had a higher proliferation rate than DPSC and BMSC. * P<0.05. Abbreviations as in Figure 1."

Figure 3

Induced stem cells in Matrigel medium showing vasculature formation A, BMSC; B, DPSC; C, SHED. D, cells without induction showed no formation of vessels; E, CD31; F, vWF; G, the total length of tube formation of induced BMSC and SHED is higher than DPSC in Matrigel medium; H, the meshes number of induced SHED is higher than DPSC and BMSC in Matrigel medium. A to C, induced stem cells in Matrigel medium under phase contrast microscope, showing vasculature formation(×100); E and F, Immunocytochemical results of induced cells, showing positive CD31 and vWF expression. * P<0.05."

Figure 4

The expression of angiogenesis-related genes after induction A, SHED; B, DPSC; C, BMSC; D, negative control; RQ, relative quantification. CD31, platelet endothelial cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VEGFR1, vascular endothelial growth factor receptor 1; VEGFR2, vascular endothelial growth factor receptor 2; vWF, von Willebrand factor. * P<0.05."

Figure 5

Effect of BMSC, DPSC and SHED on new formed vessels in CAM under phase contrast microscope A, SHED; B, DPSC; C, BMSC; D, negative control; E, statistically analysis showed no significantly difference among groups; F, statistically analysis showed the length of vessels of SHED was higher than negative control and BMSC (P<0.05). A to D, Effect of SHED, BMSC and DPSC on new formed vessels in CAM under phase contrast microscope (×40)."

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