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Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

ABSTRACT: Odontogenesis is the result of the reciprocal interactions between epithelial-mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.

SUBMITTER: Wang F

PROVIDER: S-EPMC3779316 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

Wang Feng F Wu Li-An LA Li Wentong W Yang Yuan Y Guo Feng F Gao Qingping Q Chuang Hui-Hsiu HH Shoff Lisa L Wang Wei W Chen Shuo S

In vitro cellular & developmental biology. Animal 20130629 8

Odontogenesis is the result of the reciprocal interactions between epithelial-mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the ...[more]

PMID: 23813243

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Project description:To understand the functions of secretory proteins in odontogenesis and to further the understanding of the different molecular events during odontogenesis and osteogenesis, we induced the odonto/osteogenic differentiation of stem cells from dental apical papilla (SCAPs) and bone marrow-derived stem cells (BMSCs) in vitro and compared the expression of secretory proteins during early odonto/osteogenic differentiation using high-performance liquid chromatography with tandem mass spectrometry. The results revealed significant changes by at least 50% in 139 SCAP proteins and 203 BMSC proteins during differentiation. Of these, 92 were significantly upregulated and 47 were significantly downregulated during the differentiation of SCAPs. Most of these proteins showed the same trend during the differentiation of BMSCs. Among the proteins that showed significantly changes during the differentiation of SCAPs and BMSCs, we found that transforming growth factor-?2 (TGF?2) is a key protein in the network with powerful mediation ability. TGF?2 was secreted more by SCAPs than BMSCs, was significantly upregulated during the differentiation of SCAPs and was significantly downregulated during the differentiation of BMSCs. Furthermore, the effects of recombinant human TGF?2 and TGF?1 on the odonto/osteogenic differentiation of SCAPs and BMSCs were investigated. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting data revealed that TGF?2 enhanced the odontogenic-related markers [dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP1)] and inhibited the osteogenic-related marker bone sialoprotein (BSP) in SCAPs, whereas TGF?1 enhanced the BSP expression and inhibited the DSPP and DMP1 expression at early odonto/osteogenic differentiation of SCAPs. However, in BMSCs, TGF?2 enhanced the expression of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), DSPP, and DMP1, whereas TGF?1 enhanced the expression of ALP and RUNX2, with no significant intergroup difference of DSPP at the early odonto/osteogenic differentiation of BMSCs. TGF?2 is a potentially important molecule with a distinct function in the regulation of odontogenesis and osteogenesis.

Project description:Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Project description:Bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation has been shown to occur through the canonical Wnt/?catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibit cell differentiation and promote cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with BMP2, would exhibit changes in genes/proteins associated with the Wnt/?-catenin pathway.SVF4 cells were stimulated with BMP2, and the following assays were carried out: (i) Wnt/?-catenin pathway activation assessed by western blotting, ?-catenin/transcription factor (TCF) reporter assays and expression of the lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2 (Axin2) genes; and (ii) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and by the mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp), determined by quantitative PCR after treatment with wingless-type MMTV integration site family, member 3A (WNT3A) and knockdown of ?-catenin.WNT3A induced ?-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting that the Wnt/?-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with WNT3A suppressed BMP2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, ?-catenin knockdown showed that the BMP2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous ?-catenin. WNT3A down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared with untreated cells. In contrast, BMP2 induction of Bsp transcripts occurred independently of Wnt/?-catenin signaling.These data suggest that stabilization of ?-catenin by WNT3A inhibits BMP2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although BMP2 requires endogenous Wnt/?-catenin signaling to promote cell maturation.

Dataset Information

Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

Publications

Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

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