Epiprofin Transcriptional Activation Promotes Ameloblast Induction From Mouse Induced Pluripotent Stem Cells via the BMP-Smad Signaling Axis

Abstract

The transcriptional regulation of induced pluripotent stem cells (iPSCs) holds promise for their directed differentiation into ameloblasts, which are usually lost after tooth eruption. Ameloblast differentiation is regulated by multiple signaling molecules, including bone morphogenetic proteins (BMPs). Epiprofin (Epfn), a transcription factor, is expressed in the dental epithelium, and epithelial Epfn overexpression results in ectopic ameloblast differentiation and enamel formation in mouse incisor, a striking phenotype resembling that of mice with deletion of follistatin (a BMP inhibitor). However, it remains unknown whether and how Epfn transcriptional activation promotes ameloblast induction from mouse iPSCs. Here, we generated doxycycline-inducible Epfn-expressing mouse iPSCs (Epfn-iPSCs). Ameloblasts, which are characterized by positive staining for keratin 14 and amelogenin and alizarin red S staining, were successfully derived from Epfn-iPSCs based on a stage-specific induction protocol, which involved the induction of the surface ectoderm, dental epithelial cells, and ameloblasts at stages 1, 2, and 3, respectively. Epfn activation by doxycycline at stages 2 and/or 3 decreased cell proliferation and promoted ameloblast differentiation, along with the upregulation of p-Smad1/5/8, a key regulator of the BMP-Smad signaling pathway. Gene analysis of the BMP-Smad signaling pathway-associated molecules revealed that Epfn activation decreased follistatin expression at stage 2, but increased BMP2/4/7 expression at stage 3. Perturbations in the ameloblast differentiation process were observed when the BMP-Smad signaling pathway was inhibited by a BMP receptor inhibitor (LDN-193189). Simultaneous LDN-193189 treatment and Epfn activation largely reversed the perturbations in ameloblast induction, with partial recovery of p-Smad1/5/8 expression, suggesting that Epfn activation promotes ameloblast induction from mouse iPSCs partially by upregulating BMP-Smad activity. These results reveal the potential regulatory networks between Epfn and the BMP-Smad pathway and suggest that Epfn is a promising target for inducing the differentiation of ameloblasts, which can be used in enamel and tooth regeneration.

Stiffness‐Tunable Hydrogel‐Sandwich Culture Modulates the YAP‐Mediated Mechanoresponse in Induced‐Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation

“…Immunofluorescence for cardiomyocyte‐specific marker troponin T‐C (cTnT) was performed as previously described. [ 29 ] After staining with a monoclonal anti‐cTnT antibody, anti‐mouse IgG‐Alexa488, and Hoechst, samples were observed via fluorescence or confocal microscopy. The positively stained area per EB was quantified using ImageJ software.…”

Drug delivery systems based on polyethylene glycol hydrogels for enhanced bone regeneration

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Organoids from mouse molar and incisor as new tools to study tooth-specific biology and development

“…Although in vivo experiments using genetically modified mice have provided important insights into mouse tooth development and biology, deep knowledge is still missing, particularly regarding overlap or distinctions between molars and incisors, largely due to a lack of reliable and tractable in vitro models. Previous cell culture models of mouse dental epithelium (DE) cells, including 2D immortalized, non-physiological cell lines, 2D pluripotent stem cell-derived DE-like cells, and 3D (incisor) spheroids or tooth germ aggregates, lack the important assets of tissue-derived organoids such as faithful recapitulation of tissue-specific phenotype and function and robust long-term expandability ( Nakao et al., 2007 ; Sarkar et al., 2014 ; Binder et al., 2020 ; Miao et al., 2022 ). Moreover, many of the previous models were derived from only one tooth type, thus not allowing decipherment of molar- and incisor-specific biology and development.…”