Stem Cells From Apical Papilla Research Articles

The Origin and Identification of Mesenchymal Stem Cells in Teeth: from Odontogenic to Non-odontogenic.

Mesenchymal stem cells (MSCs) in teeth have been exploited as vital seed cells for stem cell-based dental medicine. To date, several mesenchymal stem cell populations originated from odontogenic tissue have been isolated and characterized by their expression of MSC surface markers and capacity of multi-lineage differentiation, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), stem cells from apical papilla (SCAP) and so on. However, their identity in vivo remains elusive, which hinders further understanding of their application in stem cell-based tooth regeneration. Label retaining and lineage tracing analyses, which serve as gold standards for identification of stem cells in vivo, provide feasibility for identifying MSCs in teeth. In this review, we will discuss the issues of MSCs, including the origin and identification of both odontogenic and non-odontogenic MSCs, and address the role of nerve-derived Sonic hedgehog (Shh) in the regulation of MSCs in the neurovascular bundle (NVB). Based on label retaining and lineage tracing analyses, latest studies have found new populations of non-odontogenic MSCs in teeth, periarterial-derived and glial-derived, regulated by the Shh derived from nerves in the NVB, which provides a new hope for tooth regeneration.

Homeobox C10 inhibits the osteogenic differentiation potential of mesenchymal stem cells

ABSTRACTPurpose: Mesenchymal stem cells (MSCs) are a reliable cell source for tissue regeneration. However, the molecular mechanisms underlying the directed differentiation of MSCs remain unclear which impedes potential clinical applications. Recent studies have discovered that Homeobox (HOX) genes are involved in the differentiation regulation of MSCs and bone formation. In this study, we investigate the HOXC10 function in the osteogenic differentiation potential of MSCs. Materials and Methods: Stem cells from apical papilla (SCAPs) and adipose-derived stem cells (ADSCs) were used in this study. Alkaline phosphatase (ALP) activity assays, ALP staining, Alizarin red staining, quantitative calcium analysis, osteogenesis-associated gene expression, and in vivo transplantation experiments were used to study osteogenic differentiation potential. Results: Our results showed that overexpression of HOXC10 in SCAPs inhibited ALP activity and mineralization in vitro and decreased the mRNA expression of collagen alpha-1 (I) chain, bone sialoprotein, osteocalcin, and a key transcription factor, runt-related transcription factor 2, in SCAPs. Depletion of HOXC10 promoted osteogenic differentiation in SCAPs in vitro. In addition, in vivo transplantation experiments in nude mice confirmed that SCAPs osteogenesis was triggered when HOXC10 was downregulated. Furthermore, depletion of HOXC10 also enhanced osteogenic differentiation in ADSCs. Conclusions: Taken together, these results indicated that HOXC10 decreased the MSC osteogenic differentiation potential. Thus, inhibition of HOXC10 in MSCs might have the potential to improve tissue regeneration and provide insight into the mechanism underlying the directed differentiation of MSCs.

Characterization of microRNAs profiles of induced pluripotent stem cells reprogrammed from human dental pulp stem cells and stem cells from apical papilla

To compare characterization of microRNAs (miRNAs) expression profiles of induced pluripotent stem cells (iPSCs) reprogrammed from human dental pulp stem cells (DPSCs) and stem cells from apical papilla (SCAP) and screen-specific microRNA. Human DPSCs and SCAP were reprogrammed into iPSCs using a Sendai virus vector. Total RNA of human DPSCs-iPSCs and SCAP-iPSCs were extracted. miRNAs were labeled and hybridized. Slides were scanned, and images were imported into GenePix Pro 6.0 for grid alignment and data extraction. Significant differentially expressed miRNAs between the two groups were identified using fold change and P-value and were analyzed. Both human DPSCs and SCAP were successfully reprogrammed into iPSCs. Among miRNA genes analyzed by miRNA microarray, 68 were differentially expressed by more than 10-fold in DPSCs-iPSCs; 37 of these genes were up-regulated, and 31 were down-regulated. In SCAP-iPSCs, 107 genes were differentially expressed by more than 10-fold; 68 were up-regulated, and 39 were down-regulated. In both cells, only miR-302e was up-regulated, whereas 9 miRNAs were down-regulated: miR-29b-3p, miR-181b-5p, miR-4328, miR-22-5p, miR-145-5p, miR-4324, let-7b-5p, miR-181a-5p, and miR-27b-3p. Multiple miRNAs participated in reprogramming of human DPSCs and SCAP into iPSCs. Most miRNAs are related to cell cycle, transforming growth factor-β signaling pathways and epithelial-mesenchymal transition.

Characterization of microRNAs expression profiles in human dental-derived pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) technology provides a powerful means to generate and regenerate unlimited pluripotent stem cells directly from body tissue cells. Stem cells from apical papilla (SCAP) and Dental pulp stem cells (DPSCs) are present in ‘cell-rich zones’ within the dental pulp region, which are capable of regenerating pulp and dentin tissues in vivo. In this study, we investigated the difference of miRNAs expression in SCAPs and DPSCs before and after the reprogramming. Using miRNA microarray, 134 and 265 differentially expressed miRNAs in DPSCs- and SCAP-iPSCs were up-regulated compared to these before reprogramming. 117 specific miRNAs with enhanced more than 2-fold were identified in both DPSCs- and SCAP-iPSCs. Among the co-regulated miRNAs, miR-19a-3p, miR-92b-3p and miR-130b-3p showed the maximum difference, which had involvement in the cell cycle, TGF beta signaling pathway and epithelial mesenchymal transition. Using qRT-PCR analysis, the expression of miR-19a-3p, miR-92b-3p and miR-130b-3p indicated substantial increases in DPSCs-iPSCs and SCAP-iPSCs. The findings suggest that miRNAs play a part in the difference between DPSCs-iPSCs and DPSCs, as well as between SCAP-iPSCs and SCAP. The variation of miRNA expression in reprogrammed dental-derived pluripotent stem cells revealed different characteristics induced by iPSC generation.

SFRP2 enhanced the adipogenic and neuronal differentiation potentials of stem cells from apical papilla.

Dental tissue-derived mesenchymal stem cells (MSCs) are easily obtained and considered as a favorable cell source for tissue engineering, but the regulation of direct differentiation is unknown, which restricts their application. The present study investigated the effect of SFRP2, a Wnt signaling modulator, on MSC differentiation using stem cells from apical papilla (SCAPs). The cells were cultured in specific inducing medium for adipogenic, neurogenic, or chondrogenic differentiation. Over-expression of SFRP2 via retroviral infection enhanced the adipogenic and neurogenic differentiation of SCAPs. While inhibit of Wnt pathway by IWR1-endo could enhance the neurogenic differentiation potentials of SCAPs, similar with the function of SFRP2. In addition, over-expression of SFRP2 up-regulated the expression of stemness-related genes SOX2 and OCT4. Furthermore, SOX2 and OCT4 expression was significantly inhibited after lentiviral silencing of SFRP2 in SCAPs. Therefore, our results suggest that SFRP2 enhances the adipogenic and neurogenic differentiation potentials of SCAPs by up-regulating SOX2 and OCT4. Moreover, the effect of SFRP2 in neurogenic differentiation of SCAPs maybe also associated with Wnt inhibition. Our results provided useful information about the molecular mechanism underlying directed differentiation in dental tissue-derived MSCs.

Potential of human dental stem cells in repairing the complete transection of rat spinal cord

Objective. The adult spinal cord of mammals contains a certain amount of neural precursor cells, but these endogenous cells have a limited capacity for replacement of lost cells after spinal cord injury. The exogenous stem cells transplantation has become a therapeutic strategy for spinal cord repairing because of their immunomodulatory and differentiation capacity. In addition, dental stem cells originating from the cranial neural crest might be candidate cell sources for neural engineering. Approach. Human dental follicle stem cells (DFSCs), stem cells from apical papilla (SCAPs) and dental pulp stem cells (DPSCs) were isolated and identified in vitro, then green GFP-labeled stem cells with pellets were transplanted into completely transected spinal cord. The functional recovery of rats and multiple neuro-regenerative mechanisms were explored. Main results. The dental stem cells, especially DFSCs, demonstrated the potential in repairing the completely transected spinal cord and promote functional recovery after injury. The major involved mechanisms were speculated below: First, dental stem cells inhibited the expression of interleukin-1β to reduce the inflammatory response; second, they inhibited the expression of ras homolog gene family member A (RhoA) to promote neurite regeneration; third, they inhibited the sulfonylurea receptor1 (SUR-1) expression to reduce progressive hemorrhagic necrosis; lastly, parts of the transplanted cells survived and differentiated into mature neurons and oligodendrocytes but not astrocyte, which is beneficial for promoting axons growth. Significance. Dental stem cells presented remarkable tissue regenerative capability after spinal cord injury through immunomodulatory, differentiation and protection capacity.

Analysis of gene expression profiles between apical papilla tissues, stem cells from apical papilla and cell sheet to identify the key modulators in MSCs niche.

The microenvironmental niche plays the key role for maintaining the cell functions. The stem cells from apical papilla (SCAPs) are important for tooth development and regeneration. However, there is limited knowledge about the key factors in niche for maintaining the function of SCAPs. In this study, we analyse the gene expression profiles between apical papilla tissues, SCAPs and SCAPs cell sheet to identify the key genes in SCAPs niche. Microarray assays and bioinformatic analysis were performed to screen the differential genes between apical papilla tissues and SCAPs, and SCAPs and SCAPs cell sheet. Recombinant human BMP6 protein was used in SCAPs. Then CCK-8 assay, CFSE assay, alkaline phosphatase activity, alizarin red staining, quantitative calcium analysis and real-time reverse transcriptase-polymerase chain reaction were performed to investigate the cell proliferation and differentiation potentials of SCAPs. Microarray analysis found that 846 genes were up-regulated and 1203 genes were down-regulated in SCAPs compared with apical papilla tissues. While 240 genes were up-regulated and 50 genes were down-regulated in SCAPs compared to in SCAPs cell sheet. Moreover, only 31 gene expressions in apical papilla tissues were recovered in cell sheet compared with SCAPs. Bioinformatic analysis identified that TGF-β, WNT and MAPK signalling pathways may play an important role in SCAPs niche. Based on the analysis, we identified one key growth factor in niche, BMP6, which could enhance the cell proliferation, the osteo/dentinogenic, neurogenic and angiogenic differentiation potentials of SCAPs. Our results provided insight into the mechanisms of the microenvironmental niche which regulate the function of SCAPs, and identified the key candidate genes in niche to promote mesenchymal stem cells-mediated dental tissue regeneration.

IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla.

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc.

Comparison of stem cell behaviors between indigenous high and low-CD24 percentage expressing cells of stem cells from apical papilla (SCAPs).

CD24 was suggested as a marker to SCAPs and has been reported for a decade. CD24 has been shown to involve stem cell activities such as self-renewal, proliferation and differentiation. However, the percentage variations of CD24 positive cells were reported among the studies. It is possible that this variation may affect these SCAPs behaviors. In this study, the variation was confirmed. To elucidate the influence of CD24 positive cells quantity on SCAPs stem cell behaviors, the 3 cell lines with the most maximum and the least numbers of CD24 positive cells (High-CD24 and Low-CD24 group) were selected to study. Both groups expressed the same mesenchymal stem cell markers and negative to hematopoietic marker. High-CD24 group demonstrated less self-renewal capacity by lower colony-forming-unit count and pluripotency marker gene expressions. However, cell proliferation was not different. In contrast, osteogenic and adipogenic differentiation were better than Low-CD24 group. The early stage of root development demonstrated higher CD24 expressing cells than later stage. In conclusion, quantity of CD24 expressing cells influenced SCAPs self-renewal and multi-lineage differentiation but did not influence on cell proliferation. Stage of root development influenced to CD24 expressing cell numbers.

Gingiva as a new and the most accessible source of mesenchymal stem cells from the oral cavity to be used in regenerative therapies.

Since the discovery of bone marrow mesenchymal stem cells (BMMSCs), many researchers have focused their attention on new sources of mesenchymal stem cells (MSCs). Consequently, MSCs that display self-renewal capacity, multidifferentiation potential and immunomodulatory properties have been isolated from human oral tissues, including tooth, periodontal ligament, and gingiva. Oral MSCs involve dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFCs), stem cells from apical papilla (SCAP) and gingival stem cells (GMSCs). Current research on oral stem cells is expanding at an unprecedented rate. That being the case, a plethora of in vitro differentiation assays, immunodeficient animal transplantations and preclinical trials have demonstrated that these cells exhibit strong potential for both regenerative dentistry and medicine. Oral MSCs have proved their capability to repair cornea, dental pulp, periodontal, bone, cartilage, tendon, neural, muscle and endothelial tissues without neoplasm formation as well as to treat inflammatory diseases and immune disorders. This article describes the current understanding of oral MSCs and their prospective applications in cell-based therapy, tissue engineering and regenerative medicine. Special attention is placed on GMSCs as they are easily accessible and may be obtained in a convenient and minimally invasive way.

EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla

IntroductionThis study aimed to investigate the roles of ephrinB2 in stabilizing vascularlike structures generated by stem cells from apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs). MethodsHUVECs were seeded alone or with SCAPs concurrently or 12 hours later. Angiogenesis and ephrinB2 phosphorylation were assayed at different time points. Additionally, ephrinB2 expression in SCAPs and HUVECs was silenced with small interfering RNA, and vascularlike structure formation within coculture was assessed; 1 × 105 HUVECs were seeded in transwell inserts, and 6 × 105 SCAPs were plated in lower wells with or without ephrinB2-Fc. Migratory cells were stained and counted. Delayed addition of ephrinB2-Fc to the coculture of HUVECs and SCAPs was performed to evaluate the role of ephrinB2 on the stabilization of vascularlike structures. ResultsConcurrent coculture of SCAPs and HUVECs yielded significantly longer tubule lengths at 4, 8, and 12 hours (P < .05). Delayed addition of SCAPs to coculture with HUVECs resulted in vascularlike structures persisting longer than the HUVEC monoculture. Western blot confirmed that ephrinB2 phosphorylation was initiated at 0.5 hours of coculture and peaked at 1 hour. Silencing ephrinB2 expression in SCAPs and HUVECs resulted in the absence of vascularlike structures. Enhanced migration of HUVECs by SCAPs could be inhibited by ephrinB2-Fc. When ephrinB2-Fc was added at 3 hours of coculture, the vascularlike structures were stabilized for more than 12 hours as compared with 9 hours in the control group. ConclusionsEphrinB2 plays an important role in the stabilization of vascularlike structures generated by HUVECs and SCAPs.

Phosphatidylinositol 3-Kinase and Protein Kinase C Signaling Pathways Are Involved in Stromal Cell–derived Factor-1α–mediated Transmigration of Stem Cells from Apical Papilla

IntroductionPreviously, we have shown that stem cells from apical papilla (SCAPs) can be chemoattracted by stromal cell–derived factor-1α (SDF-1α). The purpose of this study was to investigate the intracellular signaling pathways involved in SDF-1α–mediated migration of SCAPs. MethodsChemotaxis assays were performed to assess the effect of phosphatidylinositol 3-kinase (PI3K) and protein kinase C (PKC) signaling pathways in the SDF-1α–mediated migration of SCAPs using inhibitors of PI3K (LY294002) or PKC (GF109203X). The Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan) was used to evaluate the effect of the inhibitors on the proliferation of SCAPs. The expression of focal adhesion–related proteins was examined by immunofluorescence staining and Western blot analysis. Phosphorylation of PI3K subunit p85 and PKC after SDF-1α induction was evaluated by Western blot. ResultsThe inhibition of PI3K or PKC signaling pathways significantly reduced SDF-1α–mediated migration of SCAPs. The inhibitors had no effect on the proliferation of SCAPs. Immunofluorescence analysis revealed that SDF-1α stimulated focal adhesion formation and stress fiber assembly in SCAPs, in addition to up-regulation of the expression of focal adhesion molecules, including p-focal adhesion kinase, p-paxillin, and vinculin. Pretreatment with PI3K or PKC inhibitors before SDF-1α induction significantly inhibited focal adhesion molecule expression. Moreover, increased phosphorylation of p85 and PKC were observed after SDF-1α stimulation, whereas these phosphorylations were down-regulated by the inhibition of PI3K or PKC signaling pathways. ConclusionsPI3K and PKC signaling pathways appear to be required for SDF-1α–mediated transmigration of SCAPs. These findings provide insights into the signaling mechanisms that underlie SDF-1α–mediated migration of SCAPs.

Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla.

Stem cells from apical papilla (SCAP) possess clear osteo-/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo-/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo-/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector-mediated gene transfection was conducted with SCAP in order to construct blank vector-transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene-transfected SCAP (SCAP-VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP-VEGF was demonstrated to exhibit increased proliferation, and SCAP-BMP2-VEGF exhibited reduced proliferation during eight days observation. SCAP-BMP2-VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP-BMP2-VEGF group exhibited a significantly greater number of ALP-positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo-/odontogenic differentiation of human SCAP.

Dentin Conditioning with Bioactive Molecule Releasing Nanoparticle System Enhances Adherence, Viability, and Differentiation of Stem Cells from Apical Papilla

IntroductionTemporal-controlled bioactive molecule (BM) releasing systems allow the delivery of appropriate concentration of BM to enhance the interaction of stem cells to dentin matrix and subsequent odontogenic differentiation in regenerative endodontics. ObjectivesThe goal of this study was to evaluate the effect of dentin conditioning with 2 variants of dexamethasone (Dex) releasing chitosan nanoparticles (CSnp), (1) Dex-CSnpI (slow releasing) and (2) Dex-CSnpII (rapid releasing), on adherence, viability, and differentiation of stem cells from apical papilla (SCAP) on root dentin exposed to endodontic irrigants. MethodsSlab-shaped dentin specimens were prepared parallel to the root canal and treated with 5.25% sodium hypochlorite (NaOCl) for 10 minutes and/or 17% EDTA for 2 minutes. Dentin was then conditioned accordingly by (1) no nanoparticle treatment, (2) CSnp, (3) Dex-CSnpI, and (4) Dex-CSnpII. The effect of nanoparticle conditioning on SCAP viability was determined by cell count and a circularity index. SCAP adherence and viability on dentin were assessed by fluorescence and scanning electron microscopy and odontogenic differentiation by immunofluorescence. ResultsSCAP on dentin treated with NaOCl alone or NaOCl as the last irrigant showed the least adherence, minimal cytoplasmic extensions, and higher circularity. SCAP adherence and viability on Dex-CSnpI and Dex-CSnpII conditioned dentin were increased and had a well-developed cytoplasmic matrix and significantly lower circularity (P < .05). SCAP cultured in Dex-CSnpII group expressed higher levels for DSPP and DMP-1 than in CSnp or Dex-CSnpI groups. ConclusionsDex-CSnpI and Dex-CSnpII conditioning of dentin enhanced SCAP adherence and viability. Temporal-controlled release of Dex from Dex-CSnpII enhanced odontogenic differentiation of SCAP. This study highlighted the ability of dentin conditioning with temporal-controlled BM releasing nanoparticles to improve the local environment in regenerative endodontics.

Effects of Sirtuin 1 on the proliferation and osteoblastic differentiation of periodontal ligament stem cells and stem cells from apical papilla.

The function of SIRT1 in the proliferation and osteoblastic differentiation of dental stem cells is unclear. The aim of this study was to assess the roles of SIRT1 in these processes using periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAPs). A defined concentration of resveratrol, an SIRT1 activator, or nicotinamide, an SIRT1 inhibitor, was administered to PDLSCs, SCAPs, and a mixed group of the two cell lines, and their effects on these processes analyzed. Cell proliferation was tested using microtitration with a tetrazolium dye (MTT). Alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteoblastic differentiation-associated genes were assessed as well. These studies demonstrated that resveratrol could promote cell proliferation of all three groups in a gradually increasing trend over time. In contrast, nicotinamide suppressed the proliferation of the three cell lines. The results also showed that the markers of osteoblastic differentiation: ALP activity, mineralization ability, and the expression levels of the osteoblastic genes ALP, osteopontin, osteocalcin, and bone sialoprotein, were enhanced in the groups with resveratrol treatment. In contrast, following addition of nicotinamide, ALP activity, mineralization ability, and the expression levels of the osteoblastic genes were down-regulated in the cells. Together, these results suggest that the SIRT1 activator and inhibitor compounds, resveratrol and nicotinamide, function at high efficiency in adjusting cell proliferation, and that SIRT1 is a powerful regulator of osteoblastic differentiation of PDLSCs and SCAPs. In addition, co-culture of the two cell lines could promote their abilities of proliferation and osteogenic differentiation.

Dental Stem Cell in Tooth Development and Advances of Adult Dental Stem Cell in Regenerative Therapies.

Stem cell-based therapies are considered as a promising treatment for many clinical usage such as tooth regeneration, bone repairation, spinal cord injury, and so on. However, the ideal stem cell for stem cell-based therapy still remains to be elucidated. In the past decades, several types of stem cells have been isolated from teeth, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), dental follicle progenitor stem cells (DFPCs) and stem cells from apical papilla (SCAP), which may be a good source for stem cell-based therapy in certain disease, especially when they origin from neural crest is considered. In this review, the specific characteristics and advantages of the adult dental stem cell population will be summarized and the molecular mechanisms of the differentiation of dental stem cell during tooth development will be also discussed.

Composition of Mineral Produced by Dental Mesenchymal Stem Cells

Mesenchymal stem cells isolated from different dental tissues have been described to have osteogenic/odontogenic-like differentiation capacity, but little attention has been paid to the biochemical composition of the material that each produces. Here, we used Raman spectroscopy to analyze the mineralized materials produced in vitro by different dental cell populations, and we compared them with the biochemical composition of native dental tissues. We show that different dental stem cell populations produce materials that differ in their mineral and matrix composition and that these differ from those of native dental tissues. In vitro, BCMP (bone chip mass population), SCAP (stem cells from apical papilla), and SHED (stem cells from human-exfoliated deciduous teeth) cells produce a more highly mineralized matrix when compared with that produced by PDL (periodontal ligament), DPA (dental pulp adult), and GF (gingival fibroblast) cells. Principal component analyses of Raman spectra further demonstrated that the crystallinity and carbonate substitution environments in the material produced by each cell type varied, with DPA cells, for example, producing a more carbonate-substituted mineral and with SCAP, SHED, and GF cells creating a less crystalline material when compared with other dental stem cells and native tissues. These variations in mineral composition reveal intrinsic differences in the various cell populations, which may in turn affect their specific clinical applications.

Effect of lipopolysaccharide on the stromal cell-derived factor-1 expression in human stem cells from apical papilla

To investigate the expression of stromal cell-derived factor-1 (SDF-1) in human stem cells from apical papilla (SCAP), and to evaluate the effect of lipopolysaccharide (LPS) on SDF-1 expression by SCAP. SCAP were isolated from dental papilla of human immature third molars. The expression of SDF-1 was evaluated by reverse transcription-PCR (RT-PCR). After SCAP being exposed to different concentrations (0.1, 1.0, 10 mg/L) of LPS for 24 and 48 h, the effect of LPS on cell proliferation and gene expression of SDF-1 was investigated by cell counting kit-8 and real-time PCR respectively, while cells without LPS stimulation were considered as negative control. LPS had no significant effect on SCAP proliferation until day 7. RT-PCR assays demonstrated that SCAP expressed SDF-1 mRNA. Different concentrations of LPS significantly promoted the SDF-1 expression in SCAP after 24 h (F = 12.102, P = 0.002) and 48 h (F = 39.054, P < 0.001) exposure, with relative gene expression ratio (experimental/control) increased to 1.4 ± 0.1, 2.2 ± 0.4, 2.3 ± 0.5 in 24 h group and 2.1 ± 0.4, 3.4 ± 0.3, 3.8 ± 0.5 in 48 h group. Isolated SCAP in cultures have the expression of SDF-1 mRNA. LPS can significantly promote the expression of SDF-1 in SCAP.

Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1–modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth

IntroductionTransforming growth factor β1 (TGF-β1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-β1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2. MethodsSCAPs were exposed to TGF-β1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho–enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA). ResultsTGF-β1 stimulated the growth and collagen content of cultured SCAPs. TGF-β1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-β1–induced cell growth and collagen content in SCAPs. TGF-β1 stimulated ALP activity at lower concentrations (0.1–1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-β1–induced ALP activity but showed little effect on 10 ng/mL TGF-β1–induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-β1. ConclusionsTGF-β1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-β1 and SCAP for engineering of pulpal regeneration and apexogenesis.