[Notch activation delayed ageing of human dental pulp cells].

OBJECTIVE: To establish model of dental pulp cells with activated Notch signaling pathway, and investigate the effect of activating Notch signaling pathway on senescence of human dental pulp cells in vitro. METHODS: Human dental pulp cells were isolated, cultured as usual, and used from the 4(th) passage. The cells were divided into the activated group and the negative control group. In the activated group, the way of coating dishes with Jagged1 protein (10 mg/L) was used to activate Notch signaling pathway. The negative control group cells received no treatment. In the 4(th), 8(th), and 10(th) passages, the expression levels of the Notch signaling pathway downstream gene Hes1 were verified by real-time quantitative PCR (RT-qPCR). The cell changes after activating Notch signaling pathway were observed at three levels: (1) The cell morphology changes were observed through invert phase contrast microscope. The cell activity was detected with MTT assay. (2) The alkaline phosphatase (ALP) expression and its activity, and senescence-associated &bgr;-galatosidase (SA-ß-Gal) expression were observed with the kit. (3) The expression changes of senescence related genes were verified using RT-qPCR. The difference between the negative control group and the activated group was analyzed using student's t test. RESULTS: The expression level of the downstream gene Hes1 of Notch signaling pathway increased after coating the dishes with Jagged1 protein, indicating the establishment of the model of dental pulp cells with activated Notch signaling pathway. Compared with the negative control group, the aging cells of the activated group appeared relatively late. In the 8(th) and 10(th) passage, the cell activity increased. In the 10th passage, ALP activity increased, but SA-ß-Gal expression decreased. p16 gene expression decreased in each passage, and p53 gene expression decreased in the 8(th) and 10(th) passages. CONCLUSION: Jagged1 could activate Notch signaling pathway effectively. Through activating Notch signaling pathway, the dental pulp cells showed a trend of senescence delay at different levels, such as cell morphology, metabolic enzyme expressions and related gene expressions.

[Effects of DAPT on proliferation of human dental pulp cells and Notch signaling pathway].

OBJECTIVE: To explore whether the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) could inhibit Notch signaling pathway in human dental pulp cells, and its effects on the proliferation ability of the cells. METHODS: Human dental pulp cells were primarily cultured from healthy premolars or wisdom teeth extracted intactly. The γ-secretase inhibitor DAPT (5 µmol/L) was added to the culture medium from passage 4 to the end. The cells of passages 4, 8 and 10 were used as check points in this study. The Real-time RT-PCR and RT2 Profiler PCR Array were applied to analyze the expression changes of Notch signaling pathway downstream genes. And the methyl thiazolyl tetrazolium (MTT) method was used to test the proliferation ability of the cells. RESULTS: After DAPT was added, Hes1 gene expression level decreased significantly in the cells of passages 4, 8 and 10 as compared with that of the same passage cells in the control group. The relative gene expression ratio (experimental/control) decreased to 0.20 in the cells of passage 10, and the difference was significant (t=33.143,P=0.001). The PCR Array results of passage 10 dental pulp cells also showed a decline of Notch signaling pathway downstream genes Hey1 and NR4A2 in the experimental group as compared with the control group. The proliferation of passages 8, and 10 experimental cells were slowed down, and the difference was significant. CONCLUSION: Notch signaling pathway of human dental pulp cells could be inhibited by DAPT effectively. The proliferation of the cells was slowed down by the effect of DAPT, and the normal life cycle of the cells was affected.

[Infiltration of CD(4)(+)CD(25)(+) T cells into the allergic asthmatic airways caused by house dust mite antigen administration.].

OBJECTIVE: To investigate whether house dust mite (HDM) could induce CD(4)(+) CD(25)(+) T cells infiltration into asthmatic airways in patients vivo. METHODS: Ten subjects with asthma underwent initial bronchoscopy during which normal saline and HDM were administered to two sublobar segments separately. The second bronchoscopy were carried out and bronchoal lavage fluid from HDM-challenged sites and saline-challenged sites were separately taken 24 h later. The differential cell counts were determined, and the absolute number of each type was calculated. At the same time, CD(3)(+), CD(4)(+) and CD(4)(+) CD(25)(+) T cells were determined by flow-cytometric analysis. We compared cellular counts in airways without and after topical instillation of HDM. RESULTS: Eosinophile granulocyte cells of broncho-alveolar fluid in the HDM-challenged sites (1.4 +/- 0.1) x 10(6)/ml are more than it in control sites (0.3 +/- 0.1) x 10(6)/ml, P < 0.003. Lymphocyte cells of BALF in the HDM-challenged sites (2.2 +/- 0.3) x 10(6)/ml are more than it in control sites (0.3 +/- 0.1) x 10(6)/ml, P < 0.001; CD(4)(+) CD(25)(+)T cells of BALF in the HDM-challenged sites (784.0 +/- 281.3) cell/microl are more than it in control sites (7.7 +/- 3.6) cell/microl, P < 0.001. CONCLUSIONS: Our findings suggest that HDM is capable of inducing CD(4)(+) CD(25)(+) T cells recruitment into non-acute mild allergic asthmatic airways.

[Observation of multi-directional differentiation capability of dental papilla cells].

OBJECTIVE: To study the types of progenitors in dental papilla cells(DPCs) and their differentiation characteristics. METHODS: DPCs of the mandibular first molars of four-day post-natal SD rats were isolated and cultured. The expression of osteocalcin (OCN) and dentin sialoprotein (DSP) were detected using DAB kit. E11 mandible primordias, tooth germs of the mandibular first molars of one-day post-natal SD rats, were isolated and transplanted under renal capsule respectively to observe whether they could survive and continue to develop. Then the pellets with and without dissociated enamel organ were transplanted under renal capsule for 24 days. RESULTS: Cultured DPCs could express positive OCN and DSP. After transplantation, the mandible primordias developed into cap stage. The continuous genesis of dentin and enamel increased the size of tooth germs. DPCs pellets formed dentin-like and bone-like structures, while pellets with enamel organ formed only dentin-like tissue adjacent to the frames. CONCLUSION: There are different progenitors residing in DPCs. The differentiation was closely related to the surrounding environment.

Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation.

The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed ßIII-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.

Human dental stem cell derived transgene-free iPSCs generate functional neurons via embryoid body-mediated and direct induction methods.

Induced pluripotent stem cells (iPSCs) give rise to neural stem/progenitor cells, serving as a good source for neural regeneration. Here, we established transgene-free (TF) iPSCs from dental stem cells (DSCs) and determined their capacity to differentiate into functional neurons in vitro. Generated TF iPSCs from stem cells of apical papilla and dental pulp stem cells underwent two methods-embryoid body-mediated and direct induction, to guide TF-DSC iPSCs along with H9 or H9 Syn-GFP (human embryonic stem cells) into functional neurons in vitro. Using the embryoid body-mediated method, early stage neural markers PAX6, SOX1, and nestin were detected by immunocytofluorescence or reverse transcription-real time polymerase chain reaction (RT-qPCR). At late stage of neural induction measured at Weeks 7 and 9, the expression levels of neuron-specific markers Nav1.6, Kv1.4, Kv4.2, synapsin, SNAP25, PSD95, GAD67, GAP43, and NSE varied between stem cells of apical papilla iPSCs and H9. For direct induction method, iPSCs were directly induced into neural stem/progenitor cells and guided to become neuron-like cells. The direct method, while simpler, showed cell detachment and death during the differentiation process. At early stage, PAX6, SOX1 and nestin were detected. At late stage of differentiation, all five genes tested, nestin, ßIII-tubulin, neurofilament medium chain, GFAP, and Nav, were positive in many cells in cultures. Both differentiation methods led to neuron-like cells in cultures exhibiting sodium and potassium currents, action potential, or spontaneous excitatory postsynaptic potential. Thus, TF-DSC iPSCs are capable of undergoing guided neurogenic differentiation into functional neurons in vitro, thereby may serve as a cell source for neural regeneration.

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.

INTRODUCTION: Previously, 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. METHODS: Chemotaxis 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. RESULTS: The 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. CONCLUSIONS: PI3K 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.

CXC Chemokine Receptor 4 Is Expressed Paravascularly in Apical Papilla and Coordinates with Stromal Cell-derived Factor-1α during Transmigration of Stem Cells from Apical Papilla.

INTRODUCTION: Stem cells from the apical papilla (SCAPs) at the apex may be attracted into the root canal space as a cell source for pulp-dentin regeneration. To test this possibility, we used in vitro transmigration models to investigate whether SCAPs can be chemoattracted by the delivery of the chemotactic cytokine stromal cell-derived factor-1α (SDF-1α). METHODS: We first examined the expression of CXC chemokine receptor 4 (CXCR4) for SDF-1α in the apical papilla and in cultured SCAPs using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and flow cytometric analyses. A standard Transwell migration assay and a 3-dimensional cell migration assay were used to analyze transmigration of SCAPs via the SDF-1α/CXCR4 axis. RESULTS: CXCR4 was expressed in the paravascular region of the apical papilla and detected in SCAP cultures. Most cultured SCAPs harbored intracellular CXCR4 (58%-99%, n = 4), whereas only a few cells had detectable CXCR4 on the cell surface (0.3%-2.34%, n = 4). Although SDF-1α had no significant effect on SCAP proliferation, it significantly promoted a higher number of migrated cells; this effect was abolished by anti-CXCR4 antibodies. Interestingly, cell surface CXCR4 on SCAPs was not detectable until after transmigration. The 3-dimensional migration assay revealed that SDF-1α significantly enhanced SCAP migration in the collagen gel. CONCLUSIONS: SCAPs can be chemoattracted via the SDF-1α/CXCR4 axis, suggesting that SDF-1α may be used clinically to induce CXCR4-expressing SCAPs in the apical papilla to transmigrate into the root canal space as an endogenous cell source for pulp regeneration.

Establishment of transgene-free induced pluripotent stem cells reprogrammed from human stem cells of apical papilla for neural differentiation.

INTRODUCTION: Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential. METHODS: A single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their pluripotency were performed. Embryoid body-mediated neural differentiation was undertaken to verify their neurogenic potential. RESULTS: TF-SCAP iPSCs were generated via a hSTEMCCA-loxP/Cre system. PCR of genomic DNA confirmed transgene excision and puromycin treatment verified the lack of pHAGE2-EF1α-Cre-IRES-PuroR integration. Transplantation of the TF-iPSCs into immunodeficient mice gave rise to teratomas containing tissues representing the three germ layers -- ectoderm (neural rosettes), mesoderm (cartilage and bone tissues) and endoderm (glandular epithelial tissues). Embryonic stem cell-associated markers TRA-1-60, TRA-2-49 and OCT4 remained positive after transgene excision. After neurogenic differentiation, cells showed neural-like morphology expressing neural markers nestin, ßIII-tubulin, NFM, NSE, NeuN, GRM1, NR1 and CNPase. CONCLUSIONS: TF-SCAP iPSCs reprogrammed from SCAP can be generated and they may be a good cell source for neurogenesis.

Involvement of Notch signalling pathway in senescence of human dental pulp cells.

OBJECTIVE: To evaluate whether the Notch signalling pathway is involved in the senescence of human dental pulp cells. METHODS: Human dental pulp cells were isolated and cultured. The Notch signalling pathway was blocked by adding DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester, γ-secretase inhibitor, 5 µmol/L) into the culture medium. Cell proliferation was evaluated by MTT assay. Cell senescence was evaluated through expression of senescenceassociated ß-galactosidase (SA-ß-Gal) using ß-galactosidase staining. RESULTS: The proliferation of the human dental pulp cells decreased dramatically after treatment with DAPT, compared with the control. SA-ß-Gal positive staining cells were seen in the DAPT group, but not in the control group. CONCLUSION: The Notch signalling pathway is involved in the senescence of human dental pulp cells.

[Acidic fibroblast growth factor for preventing motor endplate degeneration and muscular atrophy after motor nerve injury: a morphological and electrophysiological study].

OBJECTIVE: To explore measures to prevent motor endplate degeneration and muscular atrophy after motor nerve injury. METHODS: Thirty Sprague-Dawley rats were randomized into 3 equal groups. In two of the groups, the right common peroneal nerves of the rats were transected and immediately sutured with implantation of collagen gel carrier of acidic fibroblast growth factor (aFGF) or the empty carrier into the denervated tibialis anterior muscles. In the control group, the transected nerves were sutured without implantation. Six weeks after the operation, morphological and electrophysiological examinations were performed. RESULTS: In the control rats and those with empty collagen gel carrier implantation, obvious motor endplate degeneration and muscular atrophy occurred, which were not obvious in rats receiving aFGF carrier implantation. The decrement of repetitive nerve stimulation was significantly greater in the former two groups than in the latter. CONCLUSION: Implantation of collagen gel carrier of aFGF may prevent motor endplate degeneration and facilitate functional recovery of the neuromuscular junction after motor nerve injury.