Overexpression of microRNA-221 promotes the differentiation of stem cells from human exfoliated deciduous teeth to neurons through activation of Wnt/ß-catenin pathway via inhibition of CHD8.

microRNAs have been proved to function in some processes of differentiation and the effect is favorable. At present, the differentiation of stem cells is not so ideal because of the high expenses and inaccessibility. Therefore, we explored the possibility that microRNA-221 (miR-221) affects differentiation from stem cells from human deciduous tooth (SHEDs) to neurons through Wnt/ß-catenin pathway via binding to CHD8. After collection of SHEDs, differentiation from SHEDs to neurons was conducted by neurotrophic factor induction method in vitro, followed by gain- and loss-of-function experiments. Expression of neuron-related genes in SHEDs was examined by immunohistochemistry. The relationship between CHD8 and miR-221 was detected by dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to determine miR-221 expression, and the mRNA and protein expression of CHD8, Wnt/ß-catenin pathway- and neuron-related genes. Cell viability, and cell cycle and apoptosis were investigated by MTT assay and flow cytometry respectively. Dual luciferase reporter assay displayed that miR-221 targeted CHD8 and then affected the differentiation progression. Results of RT-qPCR and Western blot analysis showed that expression of Wnt/ß-catenin pathway-related genes increased significantly, CHD8 expression decreased in neuron-induced SHEDs after miR-221 overexpression or CHD8 silencing. In response to miR-221 overexpression and CHD8 silencing, cell viability and cell cycle entry were increased, and apoptosis was reduced. Moreover, overexpression of miR-221 or silencing of CHD8 elevated the expression of neuron-related genes in neuron-induced SHEDs. Taken together, upregulation of miR-221 promotes differentiation from SHEDs to neuron cells through activation of Wnt/ß-catenin pathway by binding to CHD8.

A Biobank of Stem Cells of Human Exfoliated Deciduous Teeth: Overview of Applications and Developments in Brazil.

A biobank is an organized collection of biological human material and its associated information stored for research according to regulations under institutional responsibility, without commercial purposes, being a mandatory and strategical activity for research, regenerative medicine, and innovation. Stem cells have largely been employed in research and frequently stored in biobanks, which have been used as an essential source of biological materials. Stem cells of human exfoliated deciduous teeth (SHED) are stem cells which have a high multipotency and can be easily obtained. Besides, this extremely accessible tissue has advantages with respect to storage, as the SHED obtained in childhood can be used in later life, which implies the necessity for the creation and regulation of biobanks. The proper planning for the creation of a biobank includes knowledge of the material types to be stored, requirements regarding handling and storage conditions, storage time, and room for the number of samples. Thus, this study aimed to establish an overview of the development of a SHED biobank. Ethical and legal standardization, current applications, specific orientations, and challenges for the implementation of a SHED biobank were discussed. Through this overview, we hope to encourage further studies to use SHED biobanks.

Mitochondria Regulate the Differentiation of Stem Cells from Human Exfoliated Deciduous Teeth.

Stem cells from human exfoliated deciduous teeth (SHED) are isolated from the dental pulp tissue of primary teeth and can differentiate into neuronal cells. Although SHED are a desirable type of stem cells for transplantation therapy and for the study of neurological diseases, a large part of the neuronal differentiation machinery of SHED remains unclear. Recent studies have suggested that mitochondrial activity is involved in the differentiation of stem cells. In the present work, we investigated the neuronal differentiation machinery of SHED by focusing on mitochondrial activity. During neuronal differentiation of SHED, we observed increased mitochondrial membrane potential, increased mitochondrial DNA, and elongated mitochondria. Furthermore, to examine the demand for mitochondrial activity in neuronal differentiation, we then differentiated SHED into neuronal cells in the presence of rotenone, an inhibitor of mitochondrial respiratory chain complex I, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupler, and found that neuronal differentiation was inhibited by treatment with rotenone and CCCP. These results indicated that increased mitochondrial activity was crucial for the neuronal differentiation of SHED.Key words: mitochondria, differentiation, stem cells, dental pulp, exfoliated deciduous teeth.

Transdentinal photobiostimulation of stem cells from human exfoliated primary teeth.

AIM: To evaluate the effects of infrared light-emitting diode (LED) irradiation on stem cells from human exfoliated deciduous teeth (SHEDs). METHODOLOGY: Exfoliated primary teeth were obtained (n = 3), and SHEDs obtained from the teeth were seeded on the pulpal surface of 0.2-mm-thick dentine discs produced from permanent molars. The cells were incubated for 24 h by placing the discs in plain Dulbecco's modified Eagle's medium (DMEM). The DMEM was then replaced with new culture medium formulated for odontoblast differentiation. After 12 h in the second medium, SHEDs were irradiated through the dentine discs using an infrared LED (850 nm) with a power density of 80 mW cm-2 . Energy doses (EDs) delivered to the occlusal surface of the dentine discs were 0 (control), 2 and 4 J cm-2 (n = 6). Subsequent tests were performed 72 h after irradiation. These tests included cell viability (MTT), alkaline phosphatase activity (ALP), total protein production (TP), scanning electron microscopy (SEM), as well as gene expression for ALP, Col I, DSPP and DMP-1. Data were analysed using Kruskal-Wallis and Mann-Whitney t-tests (α = 0.05). RESULTS: Both EDs (2 and 4 J cm-2 ) significantly increased cell viability and ALP activity. For TP, ALP and Col I gene expression, only the 4 J cm-2 group had significantly higher values compared to the control group. Cell morphology was not affected by irradiation. CONCLUSION: Infrared LED irradiation was capable of biostimulating SHEDs through a 0.2 mm thickness of dentine, especially at the 4 J cm-2 level.

Basic Fibroblast Growth Factor Regulates REX1 Expression Via IL-6 In Stem Cells Isolated From Human Exfoliated Deciduous Teeth.

Basic fibroblast growth factor (bFGF) regulates pluripotent marker expression and cellular differentiation in various cell types. However, the mechanism by which bFGF regulates REX1 expression in stem cells, isolated from human exfoliated deciduous teeth (SHEDs) remains unclear. The aim of the present study was to investigate the regulation of REX1 expression by bFGF in SHEDs. SHEDs were isolated and characterized. Their mRNA and protein expression levels were determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. In some experiments, chemical inhibitors were added to the culture medium to impede specific signaling pathways. Cells isolated from human exfoliated deciduous tooth dental pulp tissue expressed mesenchymal stem cell surface markers (CD44, CD73, CD90, and CD105). These cells differentiated into osteogenic and adipogenic lineages, when appropriately induced. Treating SHEDs with bFGF induced REX1 mRNA expression and this effect was attenuated by pretreatment with FGFR or Akt inhibitors. Cycloheximide pretreatment also inhibited the bFGF-induced REX1 expression, implying the involvement of intermediate molecule(s). Further, the addition of an IL-6 neutralizing antibody attenuated the bFGF-induced REX1 expression by SHEDs. In conclusion, bFGF enhanced REX1 expression by SHEDs via the FGFR and Akt signaling pathways. Moreover, IL-6 participated in the bFGF-induced REX1 expression in SHEDs. J. Cell. Biochem. 118: 1480-1488, 2017. © 2016 Wiley Periodicals, Inc.

Mass spectrometry analysis of gingival crevicular fluid in the presence of external root resorption.

INTRODUCTION: In this study, we used liquid chromatography-mass spectrometry (LC-MS) to investigate the differences in the composition of gingival crevicular fluid between resorbing deciduous molars and nonresorbing permanent teeth. The main goal was to identify novel biomarkers associated with root resorption. METHODS: Eleven children (4 boys, 7 girls) in the mixed dentition were selected to participate in this split-mouth design study, in which a deciduous second molar with radiographic evidence of root resorption served as the experimental site, and the permanent first molar on the contralateral quadrant was the control site. Gingival crevicular fluid was collected using absorbing strips. A total of 22 samples (11 root resorption, 11 control) were each analyzed with 1-dimensional LC-MS. The remaining samples were then pooled across the 11 patients and analyzed by 2-dimensional LC-MS. The output files were converted to mascot generic format, which can be used to perform protein identification with conventional search engines. RESULTS: The 2-dimensional LC-MS protocol was able to identify 2789 and 2421 proteins in the control and resorption pooled samples, respectively. In this population, we detected significantly upregulated and downregulated proteins in the teeth with root resorption. Interestingly, many of these proteins are characteristically found in exosomes. CONCLUSIONS: We identified novel proteins that might prove to be useful biomarkers of root resorption, individually or as part of a panel.

Periodontal ligament stem cells modulate root resorption of human primary teeth via Runx2 regulating RANKL/OPG system.

Physiological primary teeth exfoliation is a normal phenomenon during teeth development. However, retained primary teeth can often be observed in the patients with cleidocranial dysplasia (CCD) caused by mutation of Runx2. The potential regulative mechanism is still unknown. In the present study, periodontal ligament stem cells (PDLSCs) were derived from different resorbed stages of primary teeth and permanent teeth from normal patients and primary teeth from CCD patient. The proliferative, osteogenic and osteoclast-inductive capacities of PDLSCs from each group were detected. We demonstrated here that the proliferative ability of PDLSCs was reduced while the osteogenic and the osteoclast-inductive capacity of PDLSCs were enhanced during root resorption. The results also showed that PDLSCs from permanent teeth and CCD patient expressed low level of Runx2 and RANKL while high level of OPG. However, expression of Runx2 and RANKL were increased while expression of OPG was decreased in PDLSCs derived from resorbed teeth. Furthermore, Runx2 regulating the expression of RANKL and OPG and the osteoclast-inductive capacity of PDLSCs were confirmed by gain or loss of function assay. These data suggest that PDLSCs promote osteoclast differentiation via Runx2 upregulating RANKL and downregulating OPG, leading to enhanced root resorption that results in physiological exfoliation of primary teeth.

Mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs) induce immune modulatory profile in monocyte-derived dendritic cells.

BACKGROUND: Mesenchymal stem cells have prominent immune modulatory properties, which may have clinical applications; however their major source, bone marrow, is of limited availability. On the other hand, mesenchymal stem cells derived from human exfoliated deciduous teeth (SHEDs) are readily accessible, but their immune regulatory properties have not been completely investigated. This study was designed, therefore, to evaluate the SHEDs influence on DCs differentiation, maturation, ability to activate T cells and to expand CD4(+)Foxp3(+) T cells. METHODOLOGY/PRINCIPAL FINDINGS: The experiments were based in cellular co-culture during differentiation and maturation of monocyte derived-DCs (moDCs), with, or not, presence of SHEDs. After co-culture with SHEDs, (moDCs) presented lower expression of BDCA-1 and CD11c, in comparison to DC cultivated without SHEDs. CD40, CD80, CD83 and CD86 levels were also decreased in mature DCs (mDCs) after co-cultivation with SHEDs. To assess the ability of SHEDs-exposed moDCs to modulate T cell responses, the former were separated from SHEDs, and co-cultured with peripheral blood lymphocytes. After 5 days, the proliferation of CD4(+) and CD8(+) T cells was evaluated and found to be lower than that induced by moDCs cultivated without SHEDs. In addition, an increase in the proportion of CD4(+)Foxp3(+)IL-10(+) T cells was observed among cells stimulated by mature moDCs that were previously cultivated with SHEDs. Soluble factors released during co-cultures also showed a reduction in the pro-inflammatory cytokines (IL-2, TNF-α and IFN-γ), and an increase in the anti-inflammatory molecule IL-10. CONCLUSION/SIGNIFICANCE: This study shows that SHEDs induce an immune regulatory phenotype in moDCs cells, evidenced by changes in maturation and differentiation rates, inhibition of lymphocyte stimulation and ability to expand CD4(+)Foxp3(+) T cells. Further characterization and validation of this phenomenon could support the use of SHEDs, directly or indirectly for immune modulation in the clinical practice.

Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells.

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.

The role of dental pulp cells in resorption of deciduous teeth.

OBJECTIVE: To address the question whether dental pulp cells of exfoliating human deciduous teeth have some roles for controlling or regulating the root resorption via secreting key molecules (OPG, RANKL, CSF-1, TGFbeta, MCP-1 and Cbfa-1) in osteoclastogenesis, we used a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) method for detection of mRNA expressions for the cytokines listed. STUDY DESIGN: The dental pulps were retrieved from incisor and molar teeth in the late stage of shedding (n = 30) and from sound premolar teeth extracted for orthodontic reasons (control group; n = 30). The RT-PCR assays were used to identify targeted gene expression. RESULTS: Of the cytokines examined, RANKL and CSF-1 expressions showed significantly higher occurrence in deciduous dental pulps than in permanent teeth pulpal tissues (P < .040). CONCLUSIONS: The findings may suggest an interactive role for pulp tissue cells in the physiologic root resorption process. The cells of dental pulp may have some cytokine-producing cells which mediate monocyte-macrophage lineage to form osteo/odontoclasts, and the RANKL/RANK system might be involved in human deciduous teeth resorption.

Relationships between serial blood lead levels and exfoliated tooth dentin lead levels: models of tooth lead kinetics.

Because bones and permanent teeth accumulate lead, exfoliated deciduous teeth have been utilized as retrospective markers of cumulative exposure in epidemiological surveys. In this paper we describe four models of lead uptake by the coronal dentin of shed primary teeth, each with different assumptions and ramifications. Each model is characterized by different relationships between blood lead at several ages and tooth lead. Values observed in our cohort of normal Boston children are most compatible with models positing the largest lead contribution coming at older ages (i.e., closer to age at exfoliation). Characteristics of models incompatible with our data include (1) lead deposition only during initial calcification and (2) no loss or resorption of lead.