Abstract
Amelogenin comprises ~90% of enamel proteins; however, the involvement of Amelx transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible Amelx-expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor β1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of Amelx did not produce noticeable phenotypic changes in ameloblast differentiation, Amelx activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible Amelx transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with Amelx expression.
Highlights
IntroductionStem cell-based regenerative dentistry, with the ultimate goal of tooth regeneration, has attracted much attention in the dental field [1,2]
This article is an open access articleStem cell-based regenerative dentistry, with the ultimate goal of tooth regeneration, has attracted much attention in the dental field [1,2]
We evaluated the effects of stage-specific transcriptional activation of Amelx on ameloameloblast differentiation of Amelx-Induced pluripotent stem cells (iPSCs) by addition of Dox (Figure 5A)
Summary
Stem cell-based regenerative dentistry, with the ultimate goal of tooth regeneration, has attracted much attention in the dental field [1,2]. Tooth formation results from inductive interactions between dental epithelial cells (DECs) and mesenchymal cells [3]. DECs produce enamel, whereas dental mesenchymal cells give rise to dentin/pulp complex and periodontal tissues. Dental mesenchymal cells are retained in the dental pulp and distributed under the terms and conditions of the Creative Commons. Periodontal ligament, whereas DECs, such as ameloblasts, are lost after tooth eruption, making enamel and tooth regeneration difficult [4]. Mouse iPSCs can be differentiated into ameloblasts by ameloblast-conditioned medium or co-culture with DECs [9,10,11].
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