Project description: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.
Project description:Small non-coding microRNAs (miRNAs) are known to play crucial roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In an attempt to search for novel miRNAs that can control the fate of dental tissue-derived stem cells, we sorted side population (SP) cells, known to be enriched in stem cells, from dental pulp cells (DPCs) and periodontal ligament cells (PDLCs), and performed a miRNA array. As a result, miR-200b and miR-607 were highly expressed in SP cells, whereas miR-1260b and miR-720 was highly expressed in the differentiated main population (MP) cells. Of note, gain-and-loss of function analysis showed that miR-720 regulates the expression of pluripotency factor NANOG and DNA methyltransferases DNMT3A, DNMT3B and DNMT1 in DPCs. Knockdown of miR-720 significantly increased the levels of NANOG as well as the number of cells positive to the stem cell marker SSEA-4, but down-regulated the levels of DNMTs. miR-720 also regulated the proliferation of DPCs as determined by immunocytochemical analysis against ki-67, as well as odontogenic differentiation demonstrated by alizarin red staining, alkaline phosphatase activity and osteopontin mRNA level. Our findings identify mi-720 as a novel miRNA involved in the regulation of stem cell fate of DPCs. Two cell types (dental pulp cells(DPCs), periodontal ligament cells(PDLCs)) were sorted by FACS to isolate side population (SP) and main population (MP) cells. Then a microRNA array was performed with the SP and MP cells of DPCs and PDLCs.
Project description:Wnt regulates various cell responses. In dental pulp cells, Wnt signaling control cell proliferation, apoptosis, migration and differentiation. Here, the differential gene expression of human dental pulp stem cells treated with Wnt ligands or Wnt agonist was examined using a high throughput RNA sequencing technique. Results demonstrated that Wnt ligands or Wnt agonist altered numerous gene expression in human dental pulp stem cells.
Project description:Dental stem cells isolated from oral tissues have been shown to provide with high proliferation ability and multi-lineage differentiation potential. Although the dental stem cells possess the potential of nerve regeneration, the specific expression profile of dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) are still unclear. Here, DPSCs and PDLSCs, collected from the same tooth with 3 donors, were tested for high-throughput protein profiles by combining two-dimensional gel proteomics and TMT-based proteomics.
Project description:Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35 % of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs. Total RNAs were isolated from dental follicle cells after 48 hours of transfection with a TP53 expressions plasmid, a SP1 expressions plasmid and for control with an empty vector.
Project description:The roles of microRNAs (miRNAs) in odontogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unexplored. In this study, the underlying molecular mechanism of osteogenic differentiation in hDPSCs is investigated using miRNA profifiling.
Project description:The roles of lncRNAs and mRNAs in odontogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unexplored. In this study, the underlying molecular mechanism of osteogenic differentiation in hDPSCs is investigated using miRNA profifiling.
Project description:To evaluate the miRNA and mRNA expression profiles (miRNOME) we identified miRNAs during in vitro osteogenic differentiation of human dental pulp stem cells (DPSC). The DPSCs were cultured in the DMEM + beta-glycerol phosphate, ascorbic acid and dexamethasone for 2 dias to 21days. The microRNA or mRNA expression profiling during the differentiation process was analyzed through hybridizations with Agilent miRNA-microarray (8x15K format).
Project description:Human mesenchymal stem cells are a promising cell source for the treatment of stroke. Their primary mechanism of action occurs via neuroprotective effects by trophic factors, anti-inflammatory effects, and immunomodulation. However, the regeneration of damaged neuronal networks by cell transplantation remains still challenging. We hypothesized that cells induced to neural lineages would fit the niche, replace the lesion, and be more effective in improving symptoms compared with stem cells themselves. We investigated the characteristics of induced neural cells from human dental pulp tissue and compared the transplantation effects between these induced neural cells and uninduced dental pulp stem cells. Induced neural cells or dental pulp stem cells were intracerebrally transplanted 5 days after cerebral infarction induced by permanent middle cerebral artery occlusion in immunodeficient mice. Effects on functional recovery were also assessed through behavior testing. We used immunohistochemistry and neuron tracing to analyze the differentiation, axonal extension, and connectivity of transplanted cells to the host’s neural circuit. Transplantation of induced neural cells from human dental pulp ameliorated functional recovery after cerebral infarction compared with dental pulp stem cells. The induced neural cells comprised both neurons and glia and expressed functional voltage, and they were more related to neurogenesis in terms of transcriptomics. Induced neural cells had a higher viability than did dental pulp stem cells in hypoxic culture. We showed that induced neural cells from dental pulp tissue offer a novel therapeutic approach for recovery after cerebral infarction.
Project description:Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35 % of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.