SCF/C-Kit Signaling Induces Self-Renewal of Dental Pulp Stem Cells.

INTRODUCTION: The maintenance of a stem cell pool is imperative to enable healing processes in the dental pulp tissue throughout life. As such, knowing mechanisms underlying stem cell self-renewal is critical to understand pulp pathophysiology and pulp regeneration. The purpose of this study was to evaluate the impact of stem cell factor (SCF) signaling through its receptor tyrosine kinase (c-Kit) on the self-renewal of human dental pulp stem cells (hDPSCs). METHODS: The hDPSCs were stably transduced with lentiviral vectors expressing shRNA-c-Kit or vector control. The impact of the SCF/c-Kit axis on hDPSC self-renewal was evaluated by using a pulpsphere assay in low attachment conditions and by evaluating the expression of polycomb complex protein Bmi-1 (master regulator of self-renewal) by Western blot and flow cytometry. RESULTS: The c-Kit-silenced hDPSCs formed fewer pulpspheres when compared with hDPSCs transduced with control vector (P < .05). Evaluation of pulpsphere morphology revealed the presence of 3 distinct sphere types, ie, holospheres, merospheres, and paraspheres. Although c-Kit silencing decreased the number of holospheres compared with control cells (P < .05), it had no effect on the number of merospheres and paraspheres. Recombinant human stem cell factor (rhSCF) increased the number of holospheres (P < .05) and induced dose-dependent Bmi-1 expression in hDPSCs. As expected, the inductive capacity of rhSCF on Bmi-1 expression and fraction of Bmi-1-positive cells was inhibited when we silenced c-Kit in hDPSCs. CONCLUSIONS: These results unveiled the role of SCF/c-Kit signaling on the self-renewal of hDPSCs and suggested that this pathway enables long-term maintenance of stem cell pools in human dental pulps.

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis.

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.

The expression of periostin in dental pulp cells.

BACKGROUND AND OBJECTIVE: Dental pulp repair is a common process triggered by microbial and mechanical challenges. Matricellular modulators, such as periostin, are key for extracellular matrix stability and tissue healing. In the scope of the dental pulp, periostin expression has been reported during development and active dentinogenesis. However, the specific dental pulp cell population capable of expressing periostin in response to known regulators has not been clearly defined. Among the different relevant cell populations (i.e., stem cells, fibroblasts and pre-odontoblasts) potentially responsible for periostin expression in the dental pulp, this study aimed to determine which is the primary responder to periostin regulators. METHODS: Human dental pulp stem cells (DPSCs), human dental pulp fibroblasts (DPFs), and rat odontoblast-like cells (MDPC-23) were treated with different concentrations of TGF-ß1 or different regimens of biomechanical stimulation to evaluate periostin expression by qRT-PCR, Western blot and ELISA. Statistical analyses were performed by Student's t-test and ANOVA with Fisher's LSD post hoc tests (p ≤ 0.05). RESULTS: DPSC and MDPC-23 showed a statistically significant increase in periostin mRNA expression after exposure to TGF-ß1 for 48 h. TGF-ß1 also up-regulated periostin protein levels in DPSC. However, periostin significantly down-regulated protein expression in DPF. Different regimens of biomechanical stimulation showed different patterns in protein and mRNA periostin expression. CONCLUSIONS: Expression of periostin was identified in each of the analysed dental pulp cell lines, which can be regulated by TGF-ß1 and biomechanical stimulation. Overall, DPSCs are the most responsive cells to stimulation.

White mineral trioxide aggregate induces migration and proliferation of stem cells from the apical papilla.

INTRODUCTION: Regenerative endodontic protocols recommend white mineral trioxide aggregate (WMTA) as a capping material because of its osteoinductive properties. Stem cells from the apical papilla (SCAP) are presumed to be involved in this regenerative process, but the effects of WMTA on SCAP are largely unknown. Our hypothesis was that WMTA induces proliferation and migration of SCAP. METHODS: Here we used an unsorted population of SCAP (passages 3-5) characterized by high CD24, CD146, and Stro-1 expression. The effect of WMTA on SCAP migration was assessed by using transwells, and its effect on proliferation was determined by the WST-1 assay. Fetal bovine serum (FBS) and calcium chloride-enriched medium were used as positive controls. RESULTS: The SCAP analyzed here showed a low percentage of STRO-1+ and CD24+ cells. Both set and unset WMTA significantly increased the short-term migration of SCAP after 6 hours (P < .05), whereas calcium chloride-enriched medium did after 24 hours of exposure. Set WMTA significantly increased proliferation on days 1-5, whereas calcium-enriched medium showed a significant increase on day 7, with a significant reduction on proliferation afterwards. SCAP migration and proliferation were significantly and steadily induced by the presence of 2% and 10% FBS. CONCLUSIONS: Collectively, these data demonstrate that WMTA induced an early short-term migration and proliferation of a mixed population of stem cells from apical papilla as compared with a later and longer-term induction by calcium chloride or FBS.

Morphology of parasympathetic neurons innervating rat lingual salivary glands.

Saliva is essential for taste function and not only does saliva influence taste reception, but also taste perception initiates salivation. As a first step in investigating circuits involved in gustatory-salivary reflexes, we have studied the morphology of the rat inferior salivatory nucleus (ISN), which contains parasympathetic secretomotor neurons that control the parotid and lingual (von Ebner) salivary glands. By applying the fluorescent label Fluorogold to the cut end of the glossopharyngeal nerve, the neurons supplying only the lingual salivary glands were labeled. Confocal microscopy and three-dimensional reconstruction were used to analyze the labeled neurons in the horizontal plane to determine their morphological characteristics. Additional neurons were studied in the coronal plane to determine the influence of the plane of section on neuron morphology. Reconstructions indicated that inferior salivatory neurons extend in a rostral-caudal distribution just adjacent to the medial border of the nucleus of the solitary tract (NST). There is considerable morphological variability among neurons, with neurons having up to 6 primary dendrites and 17 dendritic segments that extend a maximum of 834 microm from the soma. However, although ISN neurons vary in the size and complexity of their dendritic trees, distributions of all measures of neuron morphology are unimodal, indicating that distinct groups of neurons are not revealed based on these measures. There is, however, variability in the orientation pattern of the dendritic trees that is not represented in either the population or mean measures. Individual neurons can be categorized with either mediolateral, rostro-caudal or no apparent preferred orientation. Comparisons of neurons in rostral, intermediate or caudal third of the ISN revealed regional differences in neuron morphology; neurons in the caudal third have significantly longer dendrites than those in the intermediate or rostral third. Thus, while ISN neurons belong to a single morphological grouping, they vary in the size and complexity of their dendritic trees, as well as having different dendritic orientations within the salivary nucleus.