Project description:Purpose: To compare the transcriptional changes of genes in dental pulp tissues with different degrees of inflammatory severity and investigate the role of RAD54B in inflamed human dental pulp cells (hDPCs) Methods: Normal, carious, and pulpitis human dental pulp tissues were collected. Total RNA extracted were subjected to RNA-sequencing and gene expression profiles were further studied by Gene Ontology (GO) and KEGG pathway analysis. DEGs (differentially expressed genes) in homologous recombination repair (HRR) were validated with qRT-PCR. The expression of RAD54B and TNF-α in human dental pulp tissues was detected by immunohistochemistry. HDPCs were cultured and RAD54B level in hDPCs was detected after LPS stimulation using western blot. CCK-8 was applied to investigate the cell proliferation of hDPCs transfected with negative control (Nc) small interfering RNA (siRNA), RAD54B siRNA, P53 siRNA or both siRNAs with or without LPS stimulation. Flow cytometry was applied to detect the cell cycle distribution, and western blot and immunofluorescence were utilized to analyze the expression of RAD54B, P53 and P21 under the above treatments. One-way and two-way ANOVA followed by LSD posttest were used for statistical analysis. Results: RNA-sequencing results identified DEGs among three groups. KEGG pathway analysis revealed enrichment of DEGs in Replication and Repair pathway. HRR and non-homologous end joining (NHEJ) components were further verified and qRT-PCR results were basically consistent with the sequencing data. RAD54B, a HRR accessory factor highly expressed in carious and pulpitis tissues compared to normal pulp, was chosen as our gene of interest. High RAD54B expression was confirmed in inflamed human dental pulp tissues and LPS-stimulated hDPCs. Upon RAD54B knockdown, P53 and P21 expressions in hDPCs were upregulated whereas the cell proliferation was significantly downregulated, accompanied with increased G2/M phase arrest. After inhibiting P53 expression in RAD54B-knockdown hDPCs, P21 expression and cell proliferation were reversed. Conclusions: Gene expression profiles of normal, carious and pulpitis human dental pulp tissues were revealed. HRR components was elucidated to function in dental pulp inflammation. Among HRR DEGs, RAD54B could regulate the cell proliferation of inflamed hDPCs via P53/P21 signaling. This research not only deepens our understanding of dental pulp inflammation but also provides a new insight to clarify the underlying mechanisms.
Project description:Dental pulp cells obtained from several donors proliferated actively in a serum-free medium STK2. The growth rate of dental pulp cells from most donors was higher in the serum-free medium than that in a medium containing 10% serum. DNA microarray analyses showed that gene expression profile of dental pulp cells grown in the serum-free medium was similar to that of cells grown in a medium containing 10% serum. However, several genes related to cell proliferation were up-regulated in dental pulp cells grown in the serum-free medium.
Project description:Human dental pulp cells (hDPCs) are one of the promising resources for regenerative medicine and tissue engineering, including derivation of induced pluripotent stem cells (iPSCs). However, our current protocol uses reagents of animal origin, mainly fetal bovine serum (FBS) with potential risk of infectious diseases and unwanted immunogenicity. This time, we designed a chemically defined protocol to isolate and maintain the growth and differentiation potentials of hDPCs.
Project description:To explore the underlying molecular mechanism of crosstalk between endothelial cells (ECs) and dermal papilla cells (DPCs), we used RNA sequencing to investigate the transcriptional changes in the human follicle dermal papilla cells (HFDPC) culture induced by conditioned medium (CM) treatment from human umbilical vein endothelial cell (HUVEC) (EC-CM).
Project description:We report the application of RNA sequencing technology for high-throughput profiling of gene expression in YAP knockdown (siYAP) adult human dental pulp stromal cells (hDPCs).To knockdown YAP in hDPCs we transfected cells with YAP specific siRNAs and the scrambled RNAs. Cells were derived from a 18~22-year-old healthy male human donor. To induce pulp inflammation we used LPS to treat cells. Total mRNA was collected using TRIzol ® (Invitrogen, CA, USA) solution. Then all the extraction procedures were conducted according to manufacture’s instructions for RNA extraction from homogenized tissues. After quality checks they were subjected to the high-throughput RNA-sequencing. Every group consists of 2 replicates which were from 2 independent experimental repeats.
Project description:Tooth pulp contains various types of cells such as endothelial cells, neurons, fibroblasts, osteoblasts, osteoclasts, and odontoblasts. As well as cells that are called "postnatal dental pulp stem cells" (DPSCs). Also, four more types of dental MSC-like populations were identified and characterized: stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from the apical papilla (SCAP) and population of dental follicle-derived progenitor cells called "dental follicle progenitor cells" (DFPCs). Most of them might be used in wide range of biomedical applications. Nevertheless, they have systematic differences in their physiology, e.g. differences in proliferative and differentiation potential. These differences are not clearly defined on molecular level yet; therefore, we performed proteomics comparison of DPSCs and PDLSCs in control and osteogenic differentiation. Donor matched DPSCs and PDLSCs were isolated from two donors by standard protocol. Then, DPSCs and PDLSCs at passage 3 were seeded into 90 mm Petri dishes (Eppendorf) and cultured in standard conditions with DMEM (Gibco) supplemented with 15% fetal bovine serum (FBS), 37°C, 5% CO2. When cells reached 90-100% confluency, the medium was changed to osteogenic medium (DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin (HyClone), 50 mg/ml ascorbic acid (Sigma Aldrich), 0.1 mM dexamethasone (Sigma Aldrich) and 10 mM b–glycerophosphate (Sigma Aldrich).
Project description:We report the application of RNA sequencing technology for high-throughput profiling of gene expression in YAP2SA-lentivirus-infected adult human dental pulp stromal cells (hDPCs).To overexpress YAP2SA in hDPCs we transfected cells with YAP2SA-lentivirus. Cells were derived from a 18~22-year-old healthy male human donor. Total mRNA was collected using TRIzol ® (Invitrogen, CA, USA) solution. Then all the extraction procedures were conducted according to manufacture’s instructions for RNA extraction from homogenized tissues. After quality checks they were subjected to the high-throughput RNA-sequencing. Every group consists of 3 replicates which were from 3 independent experimental repeats.
Project description:Using the HumanMethylation450 Beadchip, whole genomes of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and dental follicle progenitor cells (DFPCs) were compared.The DNA methylation profiles were obtained across approximately 485,512 CpGs in human odontogenic stem cells samples. Samples included DPSCs, DFPCs and PDLSCs from each 4 (12 in total) human individuals.
Project description:The purpose of this study was to examine the effects of hDPCs-CM on osteogenic differentiation of hDFCs and to compare gene expression in hDFCs in the presence or absence of hDPCs-CM Total RNAs were isolated from hDFCs in osteogenic medium for 7 days with or without hDPCs-CM.
Project description:HGF-CM increases the proliferation and migration ability of DPSC (Dental pulp stem cells) in a dosage dependent manner, and facilitates the mineralization of DPSC by upregulating odotogenic genes. Although lack of in vitro evidence, DPSC and hGF-CM could be a promising combination for pulp regeneration in the future.