Isolation and Enhancement of a Homogenous in Vitro Human Hertwig’s Epithelial Root Sheath Cell Population

Manal Farea,Abdul Rani,Kasmawati Mokhtar,Chin Lim,Ahmad Halim,Khairani Mokhtar,Nurul Abdullah,Zurairah Berahim,Adam Husein

doi:10.3390/ijms140611157

Abstract

Hertwig’s epithelial root sheath (HERS) cells play a pivotal role during root formation of the tooth and are able to form cementum-like tissue. The aim of the present study was to establish a HERS cell line for molecular and biochemical studies using a selective digestion method. Selective digestion was performed by the application of trypsin-EDTA for 2 min, which led to the detachment of fibroblast-like-cells, with the rounded cells attached to the culture plate. The HERS cells displayed a typical cuboidal/squamous-shaped appearance. Characterization of the HERS cells using immunofluorescence staining and flow cytometry analysis showed that these cells expressed pan-cytokeratin, E-cadherin, and p63 as epithelial markers. Moreover, RT-PCR confirmed that these cells expressed epithelial-related genes, such as cytokeratin 14, E-cadherin, and ΔNp63. Additionally, HERS cells showed low expression of CD44 and CD105 with absence of CD34 and amelogenin expressions. In conclusion, HERS cells have been successfully isolated using a selective digestion method, thus enabling future studies on the roles of these cells in the formation of cementum-like tissue in vitro.

Highlights

Tooth development and periodontal tissue formation are regulated by a series of reciprocal interactions between the oral epithelium and mesenchymal cells
Periodontal ligaments were digested in a mixture of collagenase and dispase, and the single-cell suspensions were seeded in keratinocyte growth medium (KGM)
The primary cultures consisted of cells with a fibroblast-like appearance and rounded/squamous epithelial cell-like appearance (Figure 1A)

Summary

Introduction

Tooth development and periodontal tissue formation are regulated by a series of reciprocal interactions between the oral epithelium and mesenchymal cells. In a study by Sonoyama et al (2007) [4], the periodontal ligament (PDL) tissue was digested with collagenase and dispase for the isolation of HERS cells prior to the single-cell suspensions being seeded and grown in a defined keratinocyte- serum-free medium (SFM). These authors reported that the HERS cells were sub-cultured only once for use in their experiments, a procedure that might not yield a homogenous population and might have compromised the accuracy of the interpretation of the results. A primary culture needs to be either sub-cultured several times until the fibroblast-like cells are eliminated [5,6,7,8] or processed with the use of a magnetic-activated cell sorting system to obtain a homogenous cell culture from the mixed cell population [9]

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Conclusion