Abstract
Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
Highlights
Stem cells are undifferentiated cells characterized by their ability for self-renewing division as well as by their capacity into differentiate to other cell types [1,2]
In order to characterize the whole population of Dental pulp cells (DPCs), the expression of stem cell surface markers was analyzed by Flow cytometry (FCM)
DPCs were sorted in order to isolate main population (MP) and side population (SP) cells, which corresponded to only a small fraction of 0.1% of the total number of sorted cells (Fig. 1A)
Summary
Stem cells are undifferentiated cells characterized by their ability for self-renewing division as well as by their capacity into differentiate to other cell types [1,2]. The identification and characterization of adult stem cells in various tissues has led to a greater understanding of development, tissue maintenance and self-renewal. MicroRNAs (miRNAs) have emerged as important regulators of stem cell maintenance and function [3,4]. Following studies showing that a set of transcription factors induces pluripotent stem cells [9], transfection of mature miRNAs has been reported to reprogram differentiated fibroblasts to pluripotency [10,11]. Previous studies have demonstrated the involvement of miRNAs (e.g., miR-30d, miR-138, miR-155, miR-18a) in the process of osteogenic or adipogenic differentiation of bone marrow stem/progenitor cells (BMSCs) [12,13,14]. MiRNAs play important roles in the determination of stem cell fate
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