Abstract
Induced pluripotent stem (iPS) cells could be induced into ameloblast-like cells by ameloblasts serum-free conditioned medium (ASF-CM), and bone morphogenetic proteins (BMPs) might be essential during the regulation of this process. The present study investigates the signal transduction that regulates the ameloblastic differentiation of iPS cells induced by ASF-CM. Mouse iPS cells were characterized and then cultured for 14 days in epithelial cell medium (control) or ASF-CM. Bone morphogenetic protein receptor II (BMPR-II) siRNA, inhibitor of Smad1/5 phosphorylation activated by activin receptor-like kinase (ALK) receptors, and inhibitors of mitogen-activated protein kinases (MAPKs) phosphorylation were used to treat the iPS cells in combination with ASF-CM. Real-time PCR, western blotting, and immunofluorescent staining were used to evaluate the expressions of ameloblast markers ameloblastin, enamelin, and cytokeratin-14. BMPR-II gene and protein levels increased markedly in ASF-CM-treated iPS cells compared with the controls, while the mRNA levels of Bmpr-Ia and Bmpr-Ib were similar between the ASF-CM and control groups. ASF-CM stimulation significantly increased the gene and protein expression of ameloblastin, enamelin and cytokeratin-14, and phosphorylated SMAD1/5, p38 MAPK, and ERK1/2 MAPK compared with the controls. Knockdown of BMPR-II and inhibition of Smad1/5 phosphorylation both could significantly reverse the increased expression of ameloblastin, enamelin, and cytokeratin-14 induced by ASF-CM, while neither inhibition of p38 nor ERK1/2 phosphorylation had significant reversing effects. We conclude that smad1/5 signaling transduction, activated by ALK receptors, regulates the ameloblastic differentiation of iPS cells induced by ameloblast-conditioned medium.
Highlights
Submitted: July 13, 2018 Accepted for publication: November 27, 2018 Last revision: January 28, 2019Stem-cell-based tissue-engineering approaches are being considered as a potential method to generate tooth tissues that closely match the physical and biomechanical properties of natural teeth.[1]
To clarify the mechanism underlying the ameloblastic differentiation of Induced pluripotent stem (iPS) induced by ameloblast serum-free conditioned medium (ASF-CM), we cultured the iPS cells for 14 days in the control medium or ASF-CM, and the gene
To further confirm the involvement of Bone morphogenetic protein receptor II (BMPR-II) in the ameloblastic differentiation of iPS cells, we pre-treated the iPS cells with a BMPR-II short interfering RNA (siRNA) before culturing them in control medium or ASF-CM, and detected the expression of ameloblast-specific genes (Ambn, ENAM and Ck14) and odontoblast-specific (Dmp-1 and dentine sialoprotein (Dsp)) genes
Summary
Submitted: July 13, 2018 Accepted for publication: November 27, 2018 Last revision: January 28, 2019Stem-cell-based tissue-engineering approaches are being considered as a potential method to generate tooth tissues that closely match the physical and biomechanical properties of natural teeth.[1]. Deciduous teeth (SHEDs), or from the apical papilla (SCAPs), are multipotent and can form structures resembling tooth tissue These dental stem cells have been applied successfully to generate dentin pulp and the periodontal ligament cement complexes.[2,3] the shortage of a patient’s own dental stem cells limits the use of this approach. Liu et al showed recently that ameloblast serum-free conditioned medium (ASF-CM) could induce ameloblast-like cells from mouse iPS cells successfully, which was evident by the positive expression of ameloblast markers AMBN and CK14. They further found that culturing mouse iPS cells in ASF-CM supplemented with bone morphogenetic protein 4 (BMP4) promoted odontogenic differentiation. The regulatory mechanism of the ameloblastic differentiation of iPS cells should be investigated further to promote the application of iPS in tooth regeneration
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