Abstract
Human dental pulp cells (hDPCs) possess the capacity to differentiate into odontoblast-like cells and generate reparative dentin in response to exogenous stimuli or injury. Ten–eleven translocation 1 (TET1) is a novel DNA methyldioxygenase that plays an important role in the promotion of DNA demethylation and transcriptional regulation in several cell lines. However, the role of TET1 in the biological functions of hDPCs is unknown. To investigate the effect of TET1 on the proliferation and odontogenic differentiation potential of hDPCs, a recombinant shRNA lentiviral vector was used to knock down TET1 expression in hDPCs. Following TET1 knockdown, TET1 was significantly downregulated at both the mRNA and protein levels. Proliferation of the hDPCs was suppressed in the TET1 knockdown groups. Alkaline phosphatase activity, the formation of mineralized nodules, and the expression levels of DSPP and DMP1 were all reduced in the TET1-knockdown hDPCs undergoing odontogenic differentiation. Based on these results, we concluded that TET1 knockdown can prevent the proliferation and odontogenic differentiation of hDPCs, which suggests that TET1 may play an important role in dental pulp repair and regeneration.
Highlights
Flow cytometry assay The stem cell phenotypic markers of Human dental pulp cells (hDPCs) were identified by flow cytometry; 105 cells were resuspended in 100 μL phosphate-buffered saline (PBS) and incubated with primary STRO-1 and CD146 antibodies at 4 °C for 1 h, using 1:100 dilutions
The results revealed the expression of STRO-1 (23.68%) and CD146 (89.96%), indicating that hDPCs contain mesenchymal progenitors
Effect of ten–eleven translocation 1 (TET1) knockdown on the proliferation of hDPCs To analyze the effect of TET1 knockdown on the proliferation of the hDPCs, growth rates were measured using the Cell Counting Kit-8 (CCK8) assay
Summary
Human dental pulp cells (hDPCs) are mesenchymal cells derived from the neural crest that exhibit plasticity and multipotency; these cells can generate reparative dentin to resist and repair injury due to infection or trauma.[1,2] A great deal of attention has been focused on the mechanisms involved in the odontogenic differentiation process for reparative dentine formation and dental pulp regeneration.[3,4]Numerous studies have proven that signal pathways play critical roles in regulating gene expression of the core transcriptional network of hDPCs, such as Wnt, transforming growth factor (TGF)-β, and Notch[1] signaling.[5,6,7] Several growth factors, including bone morphogenic proteins (BMPs) and growth and differentiation factor-5 (GDF5), have been identified as chemotactic signals to recruit progenitor cells and stimulate their proliferation and differentiation in hDPCs.[8]. Our previous study indicated that the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) promotes the odontogenic differentiation capacity of hDPCs, suggesting that DNA demethylation may provide a new mechanism for the regulation of odontogenic differentiation.[15] Recent studies have shown that ten–eleven translocation 1 (TET1), a recently discovered DNA dioxygenase, could catalyze the addition of covalent hydroxyl modifications to methylated DNA and promote DNA demethylation This reaction influences gene transcription, presumably by converting 5-methylcytosine (5mC) to 5hydroxymethylcytosine (5hmC) at specific genes.[16,17,18,19] Increasing evidence indicates that TET1 is involved in the epigenetic regulation of proliferation and differentiation in various cells such as embryonic stem cells (ESCs), adult neural progenitor cells, muscle progenitor cells, and cancer cells.[20,21,22,23,24] Previously, we reported that TET1 was expressed in hDPCs and that its expression increased during early natural differentiation and odontogenic induction.[25] the role of TET1 in the odontogenic differentiation of hDPCs remains unknown
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