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: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:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture. Comparison of the expression patterns of ECFCs that were either cultured in FBS-containing medium or in serum-free medium (five replicates each).
Project description:Derivation and expansion of human umbilical cord blood-derived endothelial colony forming cells under serum-free conditions - a transcriptome analysis. Endothelial colony forming cells (ECFCs) were isolated from term umbilical cord blood units. ECFCs were expanded under standard, fetal bovine serum (FBS) containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS-containing versus FBS-free culture.
Project description:Streptococcus mutans is a common constituent of oral biofilms and a primary etiologic agent of human dental caries. The bacteria associated with dental caries have a potent ability to produce organic acids from dietary carbohydrates and to grow and metabolize in acidic conditions. In this study, we observed supplementation with 1.5% arginine (final concentration) had inhibitory effects on the growth of S. mutans in complex and chemically defined media, particularly when cells were exposed to acid or oxidative stress. Deep-sequencing of RNA (RNA-Seq) comparing the transcriptomes of S. mutans growing in a chemically defined medium with and without 1.5% arginine in neutral and acidic pH conditions and under oxidative stress conditions revealed interesting results. The results provide new insights into the mechanisms of action by which arginine inhibits dental caries through direct adverse effects on multiple virulence-related properties of the most common human dental caries pathogen. The findings significantly enhance our understanding of the genetics and physiology of this cariogenic pathogen.
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:Adult stem cells maintain tissue integrity by continuous cellular turnover or serving progenitor cells. Human dental pulp stem cells (hDPSCs) are adult stem cells that can readily be isolated from extracted teeth, then expanded or stored for tissue repair and regeneration. However, stem cells undergo an aging process characterized by a decline in functional abilities, leading eventually to reduced organ function and delays in tissue repair. Like other stem cells, hDPSCs age and go through cellular senescence but little information is available on the mechanisms of aging or the details of the aged hDPSC phenotype. In this study, we investigated the aging phenotypes of human dental pulp and hDPSCs and attempted to rejuvenate them. In this study, hDPSCs were isolated from 23 third molars and primary cultured. Then, Flow cytometey, growth curve, dubling time, colony-forming-unit assay, cell viability after cryopreservation and in serum-free media culture, differntiation capacity for odonto-, adipo- and chondrogenesis, and RNA sequencing were analyzed. As results, the aged tooth maintained its weight of dental pulp however, the frequency of CD90+CD73+ mesenchymal stem cells inside pulp significantly decreased with donor aging. Moreover, each aged hDPSCs showed lower proliferative abilities, poorer survival rates, decreased tri-differentiation potential, and decline in metabolic activities than young hDPSCs. We also found those aged phenotypes could be recovered by cultivating aged hDPSCs in high concentration of fetal bovine serum (FBS) ex vivo and furthermore, the declined metabolic activities were restored by the culture. Here, we suggest a novel method of intervention for rejuvenating hDPSCs and these results have applications in both regenerative medicine and stem cell biology.