Transient receptor potential melastatin 4 channel controls calcium signals and dental follicle stem cell differentiation.

Elevations in the intracellular Ca(2+) concentration are a phenomena commonly observed during stem cell differentiation but cease after the process is complete. The transient receptor potential melastatin 4 (TRPM4) is an ion channel that controls Ca(2+) signals in excitable and nonexcitable cells. However, its role in stem cells remains unknown. The aim of this study was to characterize TRPM4 in rat dental follicle stem cells (DFSCs) and to determine its impact on Ca(2+) signaling and the differentiation process. We identified TRPM4 gene expression in DFSCs, but not TRPM5, a closely related channel with similar function. Perfusion of cells with increasing buffered Ca(2+) resulted in a concentration-dependent activation of currents typical for TRPM4, which were also voltage-dependent and had Na(+) conductivity. Molecular suppression with shRNA decreased channel activity and cell proliferation during osteogenesis but not adipogenesis. As a result, enhanced mineralization and phosphatase enzyme activity were observed during osteoblast formation, although DFSCs failed to differentiate into adipocytes. Furthermore, the normal agonist-induced first and secondary phases of Ca(2+) signals were transformed into a gradual and sustained increase which confirmed the channels' ability to control Ca(2+) signaling. Using whole genome microarray analysis, we identified several genes impacted by TRPM4 during DFSC differentiation. These findings suggest an inhibitory role for TRPM4 on osteogenesis while it appears to be required for adipogenesis. The data also provide a potential link between the Ca(2+) signaling pattern and gene expression during stem cell differentiation.

Expression of bone morphogenetic protein-6 in dental follicle stem cells and its effect on osteogenic differentiation.

The dental follicle (DF) plays an essential role in tooth eruption via regulation of bone resorption and bone formation. Bone morphogenetic protein-6 (BMP6) expression in the DF is coincident with bone growth in the tooth crypt. DF stem cells (DFSCs) have been shown to possess strong osteogenic capability. This study aims to determine the expression of BMP6 in DFSCs and to elucidate the role of BMP6 in the osteogenesis of DFSCs. DFSCs and their non-stem cell counterpart, DF cells (DFCs), were obtained from the DFs of rat pups. We showed that expression of BMP6 was significantly higher in the DFSCs than in the DFCs. DFSCs lost osteogenic capability during in vitro expansion, and DFSCs in late passages had reduced BMP6 expression as compared to early passages of DFSCs when they were subjected to osteogenic induction. Addition of exogenous human recombinant BMP6 (hrBMP6) to the osteogenic medium dramatically enhanced the osteogenesis of the late-passage DFSCs. Knockdown of BMP6 by short interfering RNA in the DFSCs in early passages resulted in a decrease in osteogenesis, which could be restored by addition of hrBMP6. We concluded that DFSCs need to express high levels of BMP6 to maintain their osteogenesis capability. Increased BMP6 expression seen in vivo in the DF may reflect the activation of DFSCs for osteogenic differentiation for bone growth during tooth eruption.

Requirement of alveolar bone formation for eruption of rat molars.

Tooth eruption is a localized event that requires a dental follicle (DF) to regulate the resorption of alveolar bone to form an eruption pathway. During the intra-osseous phase of eruption, the tooth moves through this pathway. The mechanism or motive force that propels the tooth through this pathway is controversial but many studies have shown that alveolar bone growth at the base of the crypt occurs during eruption. To determine if this bone growth (osteogenesis) was causal, experiments were designed in which the expression of an osteogenic gene in the DF, bone morphogenetic protein-6 (Bmp6), was inhibited by injection of the first mandibular molar of the rat with a small interfering RNA (siRNA) targeted against Bmp6. The injection was followed by electroporation to promote uptake of the siRNA. In 45 first molars injected, eruption was either delayed or completely inhibited (seven molars). In the impacted molars, an eruption pathway formed but bone growth at the base of the crypt was greatly reduced compared with the erupted first-molar controls. These studies show that alveolar bone growth at the base of the crypt is required for tooth eruption and that Bmp6 may be essential for promoting this growth.

Electroporation to deliver plasmid DNA into rat dental tissues.

BACKGROUND: Delivery of DNA into the target tissues is an important technique in gene function studies and gene therapy. Surgical treatment of tooth eruption disorders, such as impacted third molars, is a major healthcare cost. Because the dental follicle (DF) is essential for regulating tooth eruption, establishment of local gene transfer protocols is needed to determine the effect of various genes on eruption and to develop gene therapy approaches for inducing the eruption of impacted molars. METHODS: Plasmids containing lacZ reporter gene were injected into rat mandibles and then electroporated at the designated settings. Mandibles were collected 24 h after electroporation for X-gal staining to evaluate the transfection efficiency. Tissues were collected at various days post-electroporation to determine the expression of the transgene. RESULTS: For the DF, depth of injection and pulse number appear to be important. Six pulses can achieve above 80% transfection of the DF at 50 V or 120 V. For alveolar bone (AB) transfection, voltages are important, with 120 V being optimal. Regarding pulse durations, we determined that durations of 20 and 30 ms achieve the maximum transfection in AB and DF, respectively. CONCLUSIONS: The present study demonstrates for the first time the feasibility of electroporation to locally deliver plasmids into dental tissues. Parameters affecting electroporation to deliver plasmids into the dental tissues were optimized. This protocol could be used to deliver short hairpin RNA or genes of interest into the dental tissues to regulate tooth eruption. Thus, it may be possible to develop nonsurgical treatments for inducing the eruption of impacted teeth.

Draft Genome Sequence of Bacillus thuringiensis Strain UNMSM10RA, Isolated from Potato Crop Soil in Peru.

The 5.5-Mb genome sequence of Bacillus thuringiensis strain UNMSM10RA, isolated from potato crop soil, is reported in this study. The strain UNMSM10RA contains 5,347 protein-coding sequences, 105 tRNA genes, 15 rRNA genes, and 5 noncoding RNA (ncRNA) genes, with an average G+C content of 35.1%.

Transmission of a dsRNA in bell pepper and evidence that it consists of the genome of an endornavirus.

A double-stranded (ds) RNA from bell pepper (BP-dsRNA) cv Yolo Wonder was inherited maternally and paternally after crossing Yolo Wonder with Jalapeño M or Hungarian Wax pepper. Partial sequence information was obtained from two cDNA clones derived from the BP-dsRNA and based on sequence similarity was related to members of the genus Endornavirus. Clones of the BP-dsRNA hybridized with similar dsRNAs from four other pepper cultivars, suggesting that all five dsRNAs tested are related. One of the cDNA clones contained a region that had significant similarity with UDP-glucose:glycosyltransferases from fungi, bacteria, plants, and three endornaviruses. Data presented indicate that the BP-dsRNA is the genome of a distinct species of the genus Endornavirus.