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Comparative study on differentiation of cervical-loop cells and Hertwig's epithelial root sheath cells under the induction of dental follicle cells in rat.

ABSTRACT: Cervical loop cells (CLC) and Hertwig's epithelial root sheath (HERS) cells are believed to play critical roles in distinct developmental patterns between rodent incisors and molars, respectively. However, the differences in differentiation between CLC and HERS cells, and their response to inductions from dental follicle cells, remain largely unknown. In present study, CLC and HERS cells, as well as incisor dental follicle (IF) cells and molar dental follicle (MF) cells were isolated from post-natal 7-day rats. IF and MF cell derived conditioned medium (CM) was obtained for induction of CLC and HERS cells. In vitro experiments, we found that, under the induction of dental follicle cell derived CM, CLC cells maintained the epithelial polygonal-shapes and formed massive minerals, while part of HERS cells underwent shape transformation and generated granular minerals. CLC cells expressed higher enamel-forming and mineralization related genes, while HERS cells showed opposite expression patterns of BMP2, BMP4, AMBN and AMGN. In vivo, CLC cells generated enamel-like tissues while HERS cells formed cementum-periodontal ligament-like structures. Taken together, CLC and HERS cells present distinct differentiation patterns under the inductions from dental follicle cells.

SUBMITTER: Guo Y

PROVIDER: S-EPMC5916884 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Comparative study on differentiation of cervical-loop cells and Hertwig's epithelial root sheath cells under the induction of dental follicle cells in rat.

Guo Yongwen Y Guo Weihua W Chen Jie J Tian Ye Y Chen Guoqing G Tian Weidong W Bai Ding D

Scientific reports 20180425 1

Cervical loop cells (CLC) and Hertwig's epithelial root sheath (HERS) cells are believed to play critical roles in distinct developmental patterns between rodent incisors and molars, respectively. However, the differences in differentiation between CLC and HERS cells, and their response to inductions from dental follicle cells, remain largely unknown. In present study, CLC and HERS cells, as well as incisor dental follicle (IF) cells and molar dental follicle (MF) cells were isolated from post-n ...[more]

PMID: 29695816

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Project description:Background: The formation of dentin-pulp involves complex epithelial-mesenchymal interactions between Hertwig's epithelial root sheath cells (HERS) and dental papilla cells (DPCs). Earlier studies have identified some of the regulatory molecules participating in the crosstalk between HERS and DPCs and the formation of dentin-pulp. In the present study we focused on the role of HERS-secreted exosomes in DPCs and the formation of dentin-pulp. Specifically, we hypothesized that exosome-like vesicles (ELVs) might mediate the function of HERS and trigger lineage-specific differentiation of dental mesenchymal cells. To test our hypothesis, we evaluated the potential of ELVs derived from a HERS cell line (ELVs-H1) in inducing in vitro and in vivo differentiation of DPCs. Methods: ELVs-H1 were characterized using transmission electron microscopy and dynamic light scattering. The proliferation, migration, and odontoblast differentiation of DPCs after treatment with ELVs-H1, was detected by CCK8, transwell, ALP, and mineralization assays, respectively. Real time PCR and western blotting were used to detect gene and protein expression. For in vivo studies, DPC cells were mixed with collagen gel combined with or without ELVs and transplanted into the renal capsule of rats or subcutaneously into nude mice. HE staining and immunostaining were used to verify the regeneration of dentin-pulp and expression of odontoblast differentiation markers. Results: ELVs-H1 promoted the migration and proliferation of DPCs and also induced odontogenic differentiation and activation of Wnt/β-catenin signaling. ELVs-H1 also contributed to tube formation and neural differentiation in vitro. In addition, ELVs-H1 attached to the collagen gel, and were slowly released and endocytosed by DPCs, enhancing cell survival. ELVs-H1 together with DPCs triggered regeneration of dental pulp-dentin like tissue comprised of hard (reparative dentin-like tissue) and soft (blood vessels and neurons) tissue, in an in vivo tooth root slice model. Conclusion: Our data highlighted the potential of ELVs-H1 as biomimetic tools in providing a microenvironment for specific differentiation of dental mesenchymal stem cells. From a developmental perspective, these vesicles might be considered as novel mediators facilitating the epithelial-mesenchymal crosstalk. Their instructive potency might be exploited for the regeneration of dental pulp-dentin tissues.

Project description:BACKGROUND:Hertwig's epithelial root sheath (HERS) is important in guiding tooth root formation by differentiating into cementoblasts through epithelial-mesenchymal transition (EMT) and inducing odontoblastic differentiation of dental papilla through epithelial-mesenchymal interaction (EMI) during the tooth root development. Thus, HERS cells are critical for cementum and dentin formation and might be a potential cell source to achieve tooth root regeneration. However, limited availability and lifespan of primary HERS cells may represent an obstacle for biological investigation and therapeutic use of tooth tissue engineering. Therefore, we constructed, characterized, and tested the functionality of immortalized cell lines in order to produce a more readily available alternative to HERS cells. METHODS:Primary HERS cells were immortalized via infection with lentivirus vector containing the gene encoding simian virus 40 Large T Antigen (SV40LT). Immortalized HERS cell subclones were isolated using a limiting dilution method, and subclones named HERS-H1 and HERS-C2 cells were isolated. The characteristics of HERS-H1 and HERS-C2 cells, including cell proliferation, ability of epithelial-mesenchymal transformation and epithelial-mesenchymal interaction, were determined by CCK-8 assay, immunofluorescence staining, and real-time PCR. The cell differentiation into cementoblast-like cells or periodontal fibroblast-like cells was confirmed in vivo. And the inductive influence of the cell lines on dental papilla cells (DPCs) was also confirmed in vivo. RESULTS:HERS-H1 and HERS-C2 cells share some common features with primary HERS cells such as epithelial-like morphology, positive expression of CK14, E-Cadherin, and Vimentin, and undergoing EMT in response to TGF-beta. HERS-C2 cells showed the EMT characteristics and could differentiate into cementum-forming cells in vitro and generate cementum-like tissue in vivo. HERS-H1 could induce the differentiation of DPCs into odontoblasts in vitro and generation of dentin-like tissue in vivo. CONCLUSIONS:We successfully isolated and characterized novel cell lines representing two key features of HERS cells during the tooth root development and which were useful substitutes for primary HERS cells, thereby providing a biologically relevant, unlimited cell source for studies on cell biology, developmental biology, and tooth root regeneration.

Dataset Information

Comparative study on differentiation of cervical-loop cells and Hertwig's epithelial root sheath cells under the induction of dental follicle cells in rat.

Publications

Comparative study on differentiation of cervical-loop cells and Hertwig's epithelial root sheath cells under the induction of dental follicle cells in rat.

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