Development of immortalized Hertwig\u2019s epithelial root sheath cell lines for cementum and dentin regeneration

Xuebing Li,Sicheng Zhang,Weidong Tian,Guoqing Chen,Weihua Guo,Zirui Zhang

doi:10.1186/s13287-018-1106-8

Abstract

BackgroundHertwig’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.MethodsPrimary 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.ResultsHERS-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.ConclusionsWe 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.

Highlights

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
As for the mechanism involved in the HERS cells or odontoblast differentiation, our and other previous studies had shown that TGF-β1 or FGF2 could trigger the EMT of HERS cells, in which PI3K/AKT and MAPK/ERK signaling was involved [7, 8]
In HERS-H1 and HERS-C2 cell lines established in our study, TGF-β signaling was downregulated which might be responsible for the inhibition of HERS cell differentiation by inhibited EMT

Summary

Introduction

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. During the tooth root development, HERS cells can differentiate into cementum-forming cells via the process of epithelial– mesenchymal transition (EMT) [7, 8]. There have been studies speculated that ERM could develop into cementum-forming cells and reported that immortalized cell lines had already been established [7, 14, 15]. These cells are terminal products of HERS, which may not completely reflect the original characteristics of HERS cells in developing tooth. Most aspects of the biology of HERS cells are still obscure because of the lack of efficient methods to isolate stable phenotype and enough primary cells

Methods

Results

Conclusion