Yoshikazu Kamino

Characteristics and osteogenic differentiation of stem/progenitor cells in the human dental follicle analyzed by gene expression profiling.

The dental follicle is an ectomesenchymal tissue that surrounds developing tooth germ and that contains osteoblastic-lineage-committed stem/progenitor cells. We examined the osteogenic potential of human dental follicle cells (hDFC) by microarray analysis. We first compared the characteristics of hDFC with those of human bone marrow mesenchymal stem cells (hMSC). Like hMSC, hDFC expressed stem cell markers such as STRO-1 and Notch-1 and differentiated not only into the osteoblastic lineage, but also into the adipogenic lineage. We analyzed the gene expression profiles of hDFC and hMSC that were not differentiated toward the osteogenic lineage. The expression of cell markers and growth factor receptors by hDFC and hMSC was similar, whereas the expression pattern of homeobox genes differed between hDFC and hMSC. Next, we investigated gene expression in hDFC during osteogenic differentiation. Gene expression profiles were analyzed in hDFC cultured in osteogenic induction medium (OIM) or in growth medium (GM) for 3 and 10 days. Many genes whose expression was regulated under these conditions were functionally categorized as “transcription” genes. Osteogenic markers were up-regulated in hDFC during osteogenic differentiation, whereas neurogenic markers were down-regulated. The genes whose expression was regulated in hDFC during osteogenic differentiation were further analyzed by ingenuity pathway analysis and real-time polymerase chain reaction. Bone morphogenetic protein and transforming growth factor-β signaling pathways were activated in hDFC cultured in OIM for 3 days. This study indicates that the dental follicle contains stem cells and/or osteoblastic progenitor cells and is a potential cellular resource for bone regeneration therapy.

Microarray Analysis of Gene Expression Changes in Aging in Mouse Submandibular Gland

Little is known about the effect of salivary gland function during aging based on gene expression. Recently emerged DNA array technology provides a sensitive, quantitative, rapid approach to the monitoring of the global pattern of gene expression. In this study, we used high-density oligonucleotide arrays to monitor the changes of gene expression levels in the submandibular gland (SMG) by comparing adult mice with elderly adult mice. Of the 1328 genes screened, 160 genes (12.0%) showed more than two-fold changes; 154 (96.3%) of these genes, associated with transcription regulation, transport, signal transduction, and enzymes in the elderly mice, exhibited decreased expression levels. The remaining 6 genes (3.7%) in the elderly mice showed increased expression levels. In mouse SMG, analysis of these data suggests that aging may lead the gene expression to decrease than increase. Thus, DNA array technology can be a powerful tool for the identification of age-associated candidate genes for further analysis in aging.

Bone morphogenetic protein 6 stimulates mineralization in human dental follicle cells without dexamethasone

OBJECTIVE The aim of this study is to investigate the osteogenic differentiation human dental follicle cells (hDFCs) cultured with in osteogenic induction medium (OIM) without dexamethasone (DEX), and to analyze the gene expression profile during osteogenic differentiation. METHODS hDFCs, which isolated from dental follicle tissue from impacted third molar teeth, were cultured with OIM with or without DEX. Osteogenic differentiation of hDFCs was examined using Alkaline phosphatase activity and Arizarin red staining. Gene expression analysis was performed by Microarray and real time-PCR. RESULTS We showed that hDFCs have the capacity to differentiate into osteogenic lineages in osteogenic induction medium lacking DEX. We also analyzed gene expression profiling of hDFCs during osteogenic differentiation. BMP6 is up-regulated in both the presence and absence of DEX. In addition, BMP6 enhances gene expression levels of DLX-5, Runx2, and Osterix, which are transcription factors associated with osteogenic differentiation. BMP6 also stimulates phosphorylation of Smad1/5/8 which are transcription factors associated with BMP signalling at protein levels. Additionally BMP6 stimulates mineralization of hDFCs monolayers examined by Arizarin red S staining. CONCLUSION These findings suggest that hDFCs can differentiate to osteogenic lineage cells osteogenic induction medium without DEX, and BMP6 is a key gene in the osteogenic differentiation of hDFCs, and has therapeutic utility for bone regeneration and bone research.

Characterization of Rat Monoclonal Antibodies against Human β-Defensin-2

Defensins are a family of cationic antimicrobial peptides that participate in host defense. Human β-defensin (hBD)-2 has a potent bactericidal activity against a wide spectrum of microorganisms. Because human gingival epithelium is constantly exposed to a variety of microbial challenges, it is considered that hBD-2 has an important role in the protective mechanisms against oral bacterial infection. However, little is known about the production of hBD-2 in tissues of the oral cavity. Six rat monoclonal antibodies (MAbs) raised against chemically synthesized hBD-2 have been characterized. Rat MAbs were specific for the conformational epitopes on hBD-2, but not to hBD-1. To identify the epitope on hBD-2, a series of six overlapping peptides covering the hBD-2 whole sequence were synthesized and the immunoreactivities of six MAbs were examined. The FCPRRYK domain in hBD-2 was recognized by all six MAbs and suggested to be an epitope region. By immunocytochemistry, hBD-2 was localized focally in the epidermis ...