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Development of immortalized Hertwig's epithelial root sheath cell lines for cementum and dentin regeneration.

ABSTRACT: 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.

SUBMITTER: Li X

PROVIDER: S-EPMC6319004 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Development of immortalized Hertwig's epithelial root sheath cell lines for cementum and dentin regeneration.

Li Xuebing X Zhang Sicheng S Zhang Zirui Z Guo Weihua W Chen Guoqing G Tian Weidong W

Stem cell research & therapy 20190103 1

<h4>Background</h4>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 availabilit ...[more]

PMID: 30606270

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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:The objective of this study was to evaluate age related changes in physical (structure/mechanical properties) and chemical (elemental/inorganic mineral content) properties of cementum layers interfacing dentin.Human mandibular molars (N=43) were collected and sorted by age (younger=19-39, middle=40-60, older=61-81 years). The structures of primary and secondary cementum (PC, SC) types were evaluated using light and atomic force microscopy (AFM) techniques. Chemical composition of cementum layers were characterized through gravimetric analysis by estimating ash weight and concentrations of Ca, Mn, and Zn trace elements in the analytes through inductively coupled plasma mass spectroscopy. The hardness of PC and SC was determined using microindentation and site-specific reduced elastic modulus properties were determined using nanoindentation techniques.PC contained fibrous 1-3 µm wide hygroscopic radial PDL-inserts. SC illustrated PC-like structure adjacent to a multilayered architecture composing of regions that contained mineral dominant lamellae. The width of the cementum dentin junction (CDJ) decreased as measured from the cementum enamel junction (CEJ) to the tooth apex (49-21 µm), and significantly decreased with age (44-23 µm; p<0.05). The inorganic ratio defined as the ratio of post-burn to pre-burn weight increased with age within primary cementum (PC) and secondary cementum (SC). Cementum showed an increase in hardness with age (PC (0.40-0.46 GPa), SC (0.37-0.43 GPa)), while dentin showed a decreasing trend (coronal dentin (0.70-0.72 GPa); apical dentin (0.63-0.73 GPa)).The observed physicochemical changes are indicative of increased mineralization of cementum and CDJ over time. Changes in tissue properties of teeth can alter overall tooth biomechanics and in turn the entire bone-tooth complex including the periodontal ligament. This study provides baseline information about the changes in physicochemical properties of cementum with age, which can be identified as adaptive in nature.

Project description:IntroductionInvestigation of normal human thyroid function using in vitro culture systems is dependent on cells that recapitulate physiology of differentiated thyrocytes. Primary thyrocytes retain features of the native organ but have limited lifespan in culture. Immortalized thyrocytes offer an alternative if challenges maintaining phenotypic stability can be overcome to retain functional features of primary cells.Materials and methodsCI-SCREEN immortalization technology was applied to normal human thyroid tissue to generate four cell line variants. The lines were characterized for transgene integration, biomarker expression, genomic stability, and proliferation rates. Thyroid Stimulating Hormone (TSH)-dependent morphology, thyroglobulin production, thyroxine hormone synthesis, and viability were assessed using conventional 2D monolayer and 3D microtissue culture formats in huThyrEC or h7H medium.ResultsDespite differential transgene profiles, the lines had similar biomarker expression patterns and proliferation rates. In 2D culture there was no thyroxine synthesis or changes in viability, but TSH-dependent thyroglobulin production was more significant for several lines in h7H than huThyrEC medium. Comparatively, in 3D microtissues, TSH-dependent thyroglobulin induction was greater for cell lines in h7H medium. Synthesis of thyroxine in one cell line was higher than background with TSH exposure, but not significantly different than control.DiscussionImmortalization of primary human thyrocytes yielded transgenic lines of epithelial origin. When evaluated in 2D or 3D culture formats, h7H medium supported thyroglobulin production to a greater magnitude than huThyrEC medium. One cell line cultured in 3D microtissue format marginally recapitulated T4 synthesis under continuous TSH exposure.ConclusionSelect human thyroid cell lines exhibited morphological and functional features of primary thyrocytes and are a novel resource for in vitro disease modeling and toxicity testing that will enable reproducible culture models more representative of normal human thyroid function.

Project description:Tooth development is a complex process that is regulated precisely by several signalling pathways and transcription factors. GATA-binding protein 4 (GATA4) is a DNA binding transcription factor, and our previous study showed that GATA4 is a novel regulator of root development. However, it remains unclear whether GATA4 is necessary for odontoblast differentiation and dentin formation. Here, we evaluated the phenotypic changes of Wnt1-Cre;GATA4fl/fl mice. The mutant mice showed defective dentin and short root deformity. The odontoblasts lost polarity instead of exhibiting a shorter height and flattened morphology. Moreover, the expression of several molecules, such as DSPP, COL-1, DCN, and PCNA, were downregulated during mutant tooth development. In vivo, we injected lentivirus to overexpress GATA4 in mice root. The dentin formation and the expression of odonto/osteogenic markers (DSPP, COL-1, DCN) were enhanced in the GATA4 overexpression group. During the in vitro study, the ability of proliferation, migration and odonto/osteogenic differentiation was declined by GATA4 knockdown approach in human dental pulp stem cells (DPSCs). The expression of odonto/osteogenic markers (DSPP, BMP4, RUNX2, OSX, OPN, OCN) was reduced in the shGATA4 group, while overexpressing GATA4 in DPSCs promoted mineralization. Furthermore, an immunoprecipitation-mass spectrometry procedure was used to confirm the interaction between GATA4 and Fructose-1, 6-bisphosphatase 1 (FBP1). We used gain and lose-of-function to delineated the role of GATA4 in regulating FBP1 expression. Knocking down GATA4 in DPSCs resulted in decreased glucose consumption and lactate production. We used small hairpin RNA targeting FBP1 to reduce the expression of FBP1 in DPSCs, which significantly increased glucose consumption and lactate production. Together, the results suggested that GATA4 is important for root formation and odontoblast polarity, as it promotes the growth and differentiation of dental mesenchymal cells around the root and affects the glucose metabolism of DPSCs via the negative regulation of FBP1.

Dataset Information

Development of immortalized Hertwig's epithelial root sheath cell lines for cementum and dentin regeneration.

Publications

Development of immortalized Hertwig's epithelial root sheath cell lines for cementum and dentin regeneration.

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