Abstract
BackgroundTooth loss caused by caries or injuries has a negative effect on human health; thus, it is important to develop a reliable method of tooth regeneration. Research on tooth regeneration has mainly focused on mouse pluripotent stem cells, mouse embryonic stem cells, and adult stem cells from various sources in mice, whereas little has examined the differentiation of human embryonic stem (hES) cells into dental epithelium (DE) and odontogenic potential in vivo.MethodsIn this study, we induced hES cells to differentiate into dental epithelium using different concentrations of bone morphogenetic protein 4 (BMP4). With 1 pM BMP4, the hES cells differentiated into oral ectoderm (OE). These cells were then stimulated with 30 pM BMP4. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunofluorescence showed the differentiation of OE and DE. The DE generated was mixed with embryonic day 14.5 mouse dental mesenchyme (DM) and transplanted into the renal capsules of nude mice. Thirty days later, the resulting tooth-like structures were examined by micro-computed tomography and hematoxylin and eosin staining.ResultsAfter 4 days of 1 pM BMP4 stimulation, Pitx1-positive OE formed. qRT-PCR and immunofluorescence revealed that induction with 30 pM BMP4 for 2 days caused the OE to differentiate into Pitx2/Dlx2/AMBN-positive DE-like cells. These cells also expressed β-catenin and p-Smad1/5/8, which are key proteins in the Wnt/β-catenin and Bmp signaling pathways, respectively. Thirty days after in vivo transplantation, six teeth with enamel and dentin had formed on the kidney.ConclusionsThese results show that hES cells differentiated into DE after sequential stimulation with different concentrations of BMP4, and the DE thus generated showed odontogenic potential.
Highlights
Tooth loss caused by caries or injuries has a negative effect on human health; it is important to develop a reliable method of tooth regeneration
The cells were suspended in low adhesion culture plates for 2 days, and embryoid bodies (EBs) formed
More cells induced with 1 pM bone morphogenetic protein 4 (BMP4) migrated and these cells were slender and similar to epithelioid cells (Fig. 2g)
Summary
Tooth loss caused by caries or injuries has a negative effect on human health; it is important to develop a reliable method of tooth regeneration. The use of stem cell technology to produce regenerated teeth with the same morphology and function as natural teeth is the ideal way to solve the problem of tooth loss [5, 6]. Based on the theory of embryonic tooth development, regenerative teeth can be obtained by inducing stem cells to simulate the natural process of tooth development. Many members of the bone morphogenetic protein (BMP) family are involved in the development of tooth germ, including early morphogenesis, cell proliferation and differentiation, and germination [9]. We postulate that BMP4 is associated with odontogenic potential and is a key factor in the mutual induction of epithelial and mesenchyme
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