IADR Abstract Archives
Stepwise Ameloblast Induction From Induced Pluripotent Stem Cells
Objectives: Induced pluripotent stem cells (iPSCs) are promising for the development of dental epithelial cells (DECs), such as ameloblasts that are lost after tooth eruption. However, the mechanism by which ameloblasts can be induced from iPSCs using defined factors in a stepwise manner that mimics the developmental process remains largely unknown. The objective of this study was to develop a stepwise ameloblast induction protocol from iPSCs by using defined factors.
Methods: Embryoid bodies (EBs) were formed from mouse iPSCs by seesaw shaking. The EBs were seeded onto gelatin-coated plates and subjected to a stepwise induction protocol, including induction of surface ectoderm (stage 1), DECs (stage 2), and ameloblast lineage (stage 3) in sequence by optimizing several signaling molecules, such as a Nodal signaling inhibitor (SB431542) and Wnt/β-catenin activator (lithium chloride; LiCl). Cell differentiation was evaluated based on cell morphology, reverse transcription polymerase chain reaction, immunocytochemistry, western blot, and alizarin red S staining.
Results: SB431542 (5 μM) enhanced surface ectoderm differentiation at stage 1. Adjunctive use of 20 mM LiCl in addition to bone morphogenetic protein 4 and retinoic acid promoted DEC differentiation (stage 2), charactered by cobblestone appearance and keratin 14 (KRT14) expression. Attenuation of LiCl (15 mM) at stage 3 together with application of transforming growth factor β1 and epidermal growth factor resulted in ameloblast lineage with elongated cell morphology and positivity for ameloblast markers (KRT14, amelogenin, and ameloblastin) and calcium deposition. Moreover, we observed distinct stage-specific differences in cell morphology and gene expression patterns resembling the patterns present during in vivo tooth development.
Conclusions: We successfully developed a three-stage ameloblast induction strategy using a stage-specific combination of several signaling molecules. This protocol expands the current understanding of regulatory networks during ameloblast differentiation and could provide a cell sources for stem cell-based regenerative prosthodontics.
Methods: Embryoid bodies (EBs) were formed from mouse iPSCs by seesaw shaking. The EBs were seeded onto gelatin-coated plates and subjected to a stepwise induction protocol, including induction of surface ectoderm (stage 1), DECs (stage 2), and ameloblast lineage (stage 3) in sequence by optimizing several signaling molecules, such as a Nodal signaling inhibitor (SB431542) and Wnt/β-catenin activator (lithium chloride; LiCl). Cell differentiation was evaluated based on cell morphology, reverse transcription polymerase chain reaction, immunocytochemistry, western blot, and alizarin red S staining.
Results: SB431542 (5 μM) enhanced surface ectoderm differentiation at stage 1. Adjunctive use of 20 mM LiCl in addition to bone morphogenetic protein 4 and retinoic acid promoted DEC differentiation (stage 2), charactered by cobblestone appearance and keratin 14 (KRT14) expression. Attenuation of LiCl (15 mM) at stage 3 together with application of transforming growth factor β1 and epidermal growth factor resulted in ameloblast lineage with elongated cell morphology and positivity for ameloblast markers (KRT14, amelogenin, and ameloblastin) and calcium deposition. Moreover, we observed distinct stage-specific differences in cell morphology and gene expression patterns resembling the patterns present during in vivo tooth development.
Conclusions: We successfully developed a three-stage ameloblast induction strategy using a stage-specific combination of several signaling molecules. This protocol expands the current understanding of regulatory networks during ameloblast differentiation and could provide a cell sources for stem cell-based regenerative prosthodontics.