Novel strategies for expansion of tooth epithelial stem cells and ameloblast generation

Martin Binder,Leah C Biggs,Irma Thesleff,Pascal Schneider,Mark S Kronenberg,Anamaria Balic

doi:10.1038/s41598-020-60708-w

Abstract

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. Contrarily in the mouse incisors, epithelial SCs are maintained throughout life and endlessly generate ameloblasts, and thus enamel. Here we isolated Sox2-GFP+ tooth epithelial SCs which generated highly cellular spheres following a novel in vitro strategy. This system enabled analysis of SC regulation by various signaling molecules, and supported the stimulatory and inhibitory roles of Shh and Bmp, respectively; providing better insight into the heterogeneity of the SCs. Further, we generated a novel mouse reporter, Enamelin-tdTomato for identification of ameloblasts in live tissues and cells, and used it to demonstrate presence of ameloblasts in the new 3D co-culture system of dental SCs. Collectively, our results provide means of generating 3D tooth epithelium from adult SCs which can be utilized toward future generation of enamel.

Highlights

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs)
In the incisor labial cervical loops (CLs), the SCs are defined by the expression of several genes, including Sox[2], Bmi[1], Lgr[5], Gli[1] and Ptch[12,5,6,7,13], whose distinct, yet somewhat shared, expression domains indicate the heterogeneity of SCs within the niche[1]
GFP+ cells were lost between days 7 and 10, at which time cells expressing P-cadherin and Sonic hedgehog (Shh) were present in the spheres (Fig. 3E), which indicated that the Sox2-GFP+ SC populations commenced their differentiation into transit-amplifying cell (TAC)

Summary

Introduction

Enamel is secreted by ameloblasts derived from tooth epithelial stem cells (SCs). Humans cannot repair or regenerate enamel, due to early loss of tooth epithelial SCs. GFP+ cells were lost between days 7 and 10, at which time cells expressing P-cadherin and Shh were present in the spheres (Fig. 3E), which indicated that the Sox2-GFP+ SC populations commenced their differentiation into TACs. Individual addition of Shh, EDA, Noggin or Fgf[10] during the first 7 days of culture maintained high levels of GFP expression during the entire culture period (Fig. 3F–K) and had a positive effect on the sphere-formation capacity (Fig. 3L).

Results

Conclusion