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:3D cultures of primary human hepatocytes (3D PHH) are successfully used to reduce and replace the use of animal experiments in biomedical research. Yet, the initial formation of 3D PHH is highly dependent on the supplementation with fetal bovine serum (FBS). However, the molecular composition of FBS and its effects on cultured cells are poorly understood. Moreover, FBS is prone to batch-to-batch variation, immunogenic risk, and lack of adherence to the replacement, refinement and reduction (3Rs) of animal experiments. Here, we demonstrate that FBS can be fully replaced by animal-free substitutes. Specifically, we combined a previously developed animal-free substitute cocktail with a normoglycemic chemically defined culture medium. Morphological and viability evaluations along with global proteomics data, demonstrated that serum-free cultured 3D PHH have equal or superior viability and functional performance of cytochrome P450s, rendering this medium useful for long-term studies and in vitro ADMET applications. This study marks a significant advancement in the development of serum-free culture conditions for primary human hepatocytes, paving the way for more reliable and ethical in vitro studies.
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: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.