Transforming growth factor beta 3 induced odontoblast-like differentiation of stem cells from human exfoliated deciduous teeth

doi:10.3969/j.issn.2095-4344.2014.23.023

Abstract

Abstract:

BACKGROUND: Studies have reported that the superfamily of transforming growth factors exert a role in the mineralization of various stem cells, but the combination effects of transforming growth factor β3 and heparin on proliferation and mineralization ability of stem cells from human exfoliated deciduous teeth remains to be studied.

OBJECTIVE: To explore the effect of transforming growth factor β3 on odontoblast-like differentiation of stem cells from human exfoliated deciduous teeth.

METHODS: Human deciduous teeth were collected using enzyme digestion. The 3^rd passage dental pulp stem cells were incubated with 25 μg/L recombinant human transforming growth factor β3, 10 U/mL heparin or their combination. The dentin sialophosphoprotein mRNA and dentinsialoprotein expressions were detected by Q-PCR and western blot assay. Alkaline phosphatase activity was determined using alkaline phosphatase kit.

RESULTS AND CONCLUSION: Stem cells from human exfoliated deciduous teeth grew well after induction. The activity of alkaline phosphatase in the combination group was significantly higher than that in the transforming growth factor β3, heparin and control groups (P < 0.01). After combination induction, the cells were strongly positive for alizarin red staining. Results from α-PCR and western blot assay showed that the expressions of dentin sialophosphoprotein were both remarkably increased at mRNA and protein levels. In summary, stem cells from human exfoliated deciduous teeth can differentiate into odontoblast-like cells under the induction of transforming growth factor β3 plus heparin.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: tooth, deciduous, dental pulp, stem cells, transforming growth factor beta 3, heparin, dentin

CLC Number:

R394.2

Zhou Hui, Lin Jin-mei, Ren Fei, Liu Jian-ping, Zhang Jin-cai, Xu Ping-ping, Yang Qin,Chen Xiao-chun. Transforming growth factor beta 3 induced odontoblast-like differentiation of stem cells from human exfoliated deciduous teeth[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3745-3750.

Figures/Tables 1

Cell growth SHED adhered slowly. About 70% cells were adherent after 4 hours, and fundamentally spread after 48 hours. These cells were smaller and basically fusiform-shaped, possessing one or two processes. A small number of cells presented with irregular shape. Cells arranged tightly and some of them rapidly proliferated and appeared to have colony-like growth (Figure 1A). after 7 days, cells grew to 90% confluence. Immunocytochemical identification SHED was strongly positive for anti-Vimentin and STRO-1 staining in the cytoplasm (Figure 1 B, C). AKP quantitative detection The combination of 25 μg/L TGF-β3 and 10 U/mL heparin obviously promoted the activity of AKP in the SHED, significantly different from TGF-β3 and heparin alone as well as control gorup (P < 0.01; Figure 2). Odontoblast differentiation from SHED Passage 3 cells were in vitro cultured and induced for 14 days, and a small amount of brown crystals could be seen in all the groups except the control group under the microscope. At 28 days, mineralized nodules strongly positive for alizarin red staining were visible in the combination group, and bright red particles were significantly more and larger than those in the other groups. Positive cells accounted for over 90%. However, several small particles were seen in the TGF-β3 and heparin- treated groups, and the control group was negative for alizarin red staining (Figure 3). Real-time fluorescent PCR detection The combination of 25 μg/L TGF-β3 and 10 U/mL heparin obviously promoted the mRNA expression of dentin sialophosphoprotein in the SHED, significantly different from TGF-β3 and heparin alone as well as control group (P < 0.01; Figure 4). Western blot assay of the protein expression of dentin sialophosphoprotein Western blot assay showed that 25 μg/L TGF-β3 combined with 10 U/mL heparin obviously increased the protein expression of dentin sialophosphoprotein in the SHED, significantly different from TGF-β3 and heparin alone as well as control group (P < 0.01; Figure 5)."

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