[EO-12] Effects of Controlled Overexpression of AmeloD on Ameloblast Differentiation of Induced Pluripotent Stem Cells
[Abstract]
[Objectives]
Induced pluripotent stem cells (iPSCs) are a promising cell source for bioengineered tooth germ in prosthetic dentistry. AmeloD, a newly identified transcriptional factor that is highly expressed in inner enamel epithelial cells, is linked to promoting ameloblast differentiation 1). However, the mechanism behind its downstream remains unknown. The present study aimed to elucidate the role of AmeloD in enamel formation. The specific objectives were to establish doxycycline (Dox)-inducible AmeloD-expressing mouse iPSCs (AmeloD-iPSCs) and to investigate the effects of controlled transcriptional regulation of AmeloD on ameloblast differentiation of iPSCs using a stepwise ameloblast induction protocol.
[Methods]
AmeloD-iPSCs were generated using the Dox-inducible AmeloD-expressing piggyBac transposon vector with GFP reporter. The optimal Dox concentration was evaluated after 24h by examining GFP and AmeloD mRNA expression. The pluripotency of transfected iPSCs was confirmed using ALP staining, immunofluorescence staining, and semi-quantitative RT-PCR analysis. AmeloD-iPSCs were subjected to the stepwise ameloblast induction protocol, with AmeloD overexpression at each stage: surface ectoderm (stage1), dental epithelial cells (stage2), and ameloblasts (stage3). The expression of dental epithelial and ameloblast marker genes was assessed using real-time RT-PCR analysis. Alizarin Red S staining was conducted to assess calcification.
[Results and Discussion]
AmeloD-iPSCs were successfully established and maintained pluripotency as indicated by the expression of sox2, oct3/4, and nanog, as well as positive staining for ALP and Nanog. An optimal Dox concentration for inducing AmeloD expression was 1 μg/mL, as shown by the peak expression of AmeloD and GFP. During ameloblast induction, the overexpression of AmeloD in stages 1 and 2 promoted calcification and significantly increased the expression of ameloblast-related marker genes Klk4 and Mmp20. It also showed that cleavage of enamel matrix proteins and mineral replacement may be because of Klk4 and Mmp20 gene expression as a result of AmeloD overexpression in stages 1 and 2. These results suggest that AmeloD is critical in the calcification process of ameloblasts during ameloblast induction. This finding sheds light on AmeloD roles and ameloblast differentiation.
[Reference]
1) Jia L, Chiba Y, Saito K et al. The tooth-specific basic helix-loop-helix factor AmeloD promotes differentiation of ameloblasts. J Cell Physiol 2022;237(2):1597-1606.
[Objectives]
Induced pluripotent stem cells (iPSCs) are a promising cell source for bioengineered tooth germ in prosthetic dentistry. AmeloD, a newly identified transcriptional factor that is highly expressed in inner enamel epithelial cells, is linked to promoting ameloblast differentiation 1). However, the mechanism behind its downstream remains unknown. The present study aimed to elucidate the role of AmeloD in enamel formation. The specific objectives were to establish doxycycline (Dox)-inducible AmeloD-expressing mouse iPSCs (AmeloD-iPSCs) and to investigate the effects of controlled transcriptional regulation of AmeloD on ameloblast differentiation of iPSCs using a stepwise ameloblast induction protocol.
[Methods]
AmeloD-iPSCs were generated using the Dox-inducible AmeloD-expressing piggyBac transposon vector with GFP reporter. The optimal Dox concentration was evaluated after 24h by examining GFP and AmeloD mRNA expression. The pluripotency of transfected iPSCs was confirmed using ALP staining, immunofluorescence staining, and semi-quantitative RT-PCR analysis. AmeloD-iPSCs were subjected to the stepwise ameloblast induction protocol, with AmeloD overexpression at each stage: surface ectoderm (stage1), dental epithelial cells (stage2), and ameloblasts (stage3). The expression of dental epithelial and ameloblast marker genes was assessed using real-time RT-PCR analysis. Alizarin Red S staining was conducted to assess calcification.
[Results and Discussion]
AmeloD-iPSCs were successfully established and maintained pluripotency as indicated by the expression of sox2, oct3/4, and nanog, as well as positive staining for ALP and Nanog. An optimal Dox concentration for inducing AmeloD expression was 1 μg/mL, as shown by the peak expression of AmeloD and GFP. During ameloblast induction, the overexpression of AmeloD in stages 1 and 2 promoted calcification and significantly increased the expression of ameloblast-related marker genes Klk4 and Mmp20. It also showed that cleavage of enamel matrix proteins and mineral replacement may be because of Klk4 and Mmp20 gene expression as a result of AmeloD overexpression in stages 1 and 2. These results suggest that AmeloD is critical in the calcification process of ameloblasts during ameloblast induction. This finding sheds light on AmeloD roles and ameloblast differentiation.
[Reference]
1) Jia L, Chiba Y, Saito K et al. The tooth-specific basic helix-loop-helix factor AmeloD promotes differentiation of ameloblasts. J Cell Physiol 2022;237(2):1597-1606.