Induction of hESCs into Ameloblast Lineage Cells

Adult human and rodent dental mesenchymal cells have been shown to form dentin tissues. However, the lack of a viable source of human epithelial cells, which can differentiate into enamel forming ameloblasts, is potentially a limiting factor in developing strategies for total tooth regeneration. Objectives: To determine the odontogenic inductive potential of human fetal mesenchymal cells (FDPC) and adult dental mesenchymal cells (ADPC) in differentiating human embryonic stem cells (hESCs) to ameloblast lineage cells. Methods: hESCs were induced into an epithelial fate (hESC-derived epithelial cells, ES-ECs) by administration of BMP4 and retinoic acid. ES-ECs were then co-cultured with either FDPC or ADPC in MatrigelTM for 8 weeks. Cells were retrieved from MatrigelTM and gene expression was quantified by qPCR. ES-ECs/mesenchymal cells were transplanted into nude mouse renal capsules. Transplants were recovered for morphological and immunochemical studies 8 weeks post transplantation. Results: After induction, hESCs adopted an epithelial phenotype with significant up-regulation of cytokeratin 14 but negative for amelogenin. After 3-D co-culture with FDPC, ES-ECs polarized. Amelogenin expression was detected by immunofluorescene and qPCR. ADPC had no similar effect. qPCR analysis showed that FDPC had higher FGF3 and Wnt5a expression levels compared to ADPC. After in vivo transplantation, ES-ECs co-cultured with FDPC secreted extracellular matrix and formed tooth bud-like structures with polarized cells and up-regulation of amelogenin. Conclusions: In contrast to ADPC, FDPC are capable of inducing ES-ECs differentiation into amelogenin expressing cells, characteristic of ameloblast lineage cells. Further studies to investigate the role of FGF3 and Wnt5a in promoting dental epithelial cells differentiation are in progress.

This study was supported by NIH/NIDCR grants R03 DE019507-02 and R21 DE018633.