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Biomedical subjects

Motokazu Tsuneto

Publications and source records attributed to Motokazu Tsuneto.

8 recordsLinked to original sources
PubMed2006

Potential of dental mesenchymal cells in developing teeth.

The tooth, composed of dentin and enamel, develops through epithelium-mesenchyme interactions. Neural crest (NC) cells contribute to the dental mesenchyme in the developing tooth and differentiate into dentin-secreting odontoblasts. NC cells are known to differentiate into chondrocytes and osteoblasts in the craniofacial region. However, it is not clear whether the dental mesenchymal cells in the developing tooth possess the potential to differentiate into a lineage(s) other than the odontoblast lineage. In this study, we prepared mesenchymal cells from E13.5 tooth germ cells and assessed their potential for differentiation in culture. They differentiated into odontoblasts, chondrocyte-like cells, and osteoblast-like cells. Their derivation was confirmed by tracing NC-derived cells as LacZ(+) cells using P0-Cre/Rosa26R mice. Using the flow cytometry-fluorescent di-beta-D-galactosidase system, which makes it possible to detect LacZ(+) cells as living cells, cell surface molecules of dental mesenchymal cells were characterized. Large number of LacZ(+) NC-derived cells expressed platelet-derived growth factor receptor alpha and integrins. Taken together, these results suggest that NC-derived cells with the potential to differentiate into chondrocyte-like and osteoblast-like cells are present in the developing tooth, and these cells may contribute to tooth organogenesis.

Animals↗
PubMed2005

Ascorbic acid promotes osteoclastogenesis from embryonic stem cells.

Ascorbic acid (AA) is known to regulate cell differentiation; however, the effects of AA on osteoclastogenesis, especially on its early stages, remain unclear. To examine the effects of AA throughout the process of osteoclast development, we established a culture system in which tartrate-resistant acid phosphate (TRAP)-positive osteoclasts were induced from embryonic stem cells without stromal cell lines. In this culture system, the number of TRAP-positive cells was strongly increased by the addition of AA during the development of osteoclast precursors, and reducing agents, 2-mercaptoethanol, monothioglycerol, and dithiothreitol, failed to substitute for AA. The effect of AA was stronger when it was added during the initial 4 days during the development of mesodermal cells than when it was added during the last 4 days. On day 4 of the culture period, AA increased the total cell recovery and frequency of osteoclast precursors. Magnetic cell sorting using anti-Flk-1 antibody enriched osteoclast precursors on day 4, and the proportion of Flk-1-positive cells but not that of platelet-derived growth factor receptor alpha-positive cells was increased by the addition of AA. These results suggest that AA might promote osteoclastogenesis of ES cells through increasing Flk-1-positive cells, which then give rise to osteoclast precursors.

Animals↗
PubMed2005

Enforced expression of PU.1 rescues osteoclastogenesis from embryonic stem cells lacking Tal-1.

Transcription factor T-cell acute lymphocytic leukemia 1 (Tal-1) is essential for the specification of hematopoietic development. Mice lacking Tal1 fail to generate any hematopoietic precursors. Using our co-culture system with stromal cells, we demonstrate that enforced expression of the transcription factor PU.1 under tetracycline control in Tal1-null embryonic stem (ES) cells rescues the development of osteoclasts and macrophage-like phagocytes. It was low efficiency compared with wild-type ES cells; other hematopoietic lineage cells of granulocytes, B cells, mast cells, megakaryocytes, and erythroid cells were not generated. Osteoclasts developed in this culture were multinucleated and competent for bone resorption. Their development depended on macrophage colony-stimulating factor and receptor activator of nuclear factor kappaB ligand. The majority of cells with the potential to differentiate into osteoclasts expressed fetal liver kinase 1 (Flk-1) and could be isolated using anti-Flk-1 antibody. These results suggest that the expression of PU.1 is a critical event for osteoclastogenesis and that Tal-1 may lie upstream of PU.1 in a regulatory hierarchy during osteoclastogenesis.

Basic Helix-Loop-Helix Proteins↗
PubMed2004

Lipopolysaccharide-induced osteoclastogenesis in Src homology 2-domain phosphatase-1-deficient viable motheaten mice.

Osteoclasts are hemopoietic cells that participate in bone resorption and remodeling. Receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are critical for development of osteoclasts. The Toll-like receptor (TLR) family shares some of the downstream signaling with RANK. The TLR4 ligand, lipopolysaccharide (LPS), is reported to accelerate bone lysis; however, signaling via TLRs has never been reported to induce osteoclastogenesis without RANKL. In this study we showed that significant numbers of mature osteoclasts were generated from protein tyrosine phosphatase Src homology 2-domain phosphatase-1-defective Hcph(me-v)/Hcph(me-v) (me(v)/me(v)) bone marrow cells in the presence of M-CSF and LPS without addition of RANKL in culture. This M-CSF plus LPS-induced osteoclastogenesis was not inhibited by an anti-TNFalpha antagonistic antibody or by osteoprotegerin, a decoy receptor for RANKL. The replacement of RANKL by TLR ligands only occurred with LPS. Other ligands, a peptidoglycan for TLR2 or an unmethylated CpG oligonucleotide for TLR9, did not support osteoclast generation. The osteoclast precursors as well as RANKL-responsive osteoclast precursors were present in the Kit-positive cell-enriched fraction of bone marrow cells. Although me(v)/me(v) bone marrow cells required a comparable concentration of RANKL or TNFalpha as wild-type cells for the initiation of osteoclastogenesis, the numbers of multinucleated osteoclasts in me(v)/me(v) bone marrow cultures were significantly increased by the equivalent dose of RANKL or TNFalpha in the presence of M-CSF. These results indicate that a defect of Src homology 2-domain phosphatase-1 function not only accelerates physiological osteoclast development by RANKL/RANK, but also acquires a novel pathway for osteoclastogenesis by LPS.

Animals↗
PubMed2003

Distinct osteoclast precursors in the bone marrow and extramedullary organs characterized by responsiveness to Toll-like receptor ligands and TNF-alpha.

Osteoclasts are derived from hemopoietic stem cells and play critical roles in bone resorption and remodeling. Multinucleated osteoclasts are attached tightly to bone matrix, whereas precursor cells with the potential to differentiate into osteoclasts in culture are widely distributed. In this study, we assessed the characteristics of osteoclast precursors in bone marrow (BM) and in extramedullary organs as indicated by their responsiveness to ligands for Toll-like receptors (TLRs) and to TNF-alpha. Development of osteoclasts from precursor cells in the BM was inhibited by CpG oligonucleotides, a ligand for TLR9, but not by LPS, a ligand for TLR4. BM osteoclasts were induced by TNF-alpha as well as receptor activator of NF-kappaB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-alpha or receptor activator of NF-kappaB ligand. However, splenic osteoclastogenesis was inhibited by both LPS and CpG. Osteoclastogenesis from peritoneal precursors was inhibited by not only these TLR ligands but also TNF-alpha. The effects of peptidoglycan, a ligand for TLR2, were similar to those of LPS. BM cells precultured with M-CSF were characterized with intermediate characteristics between those of splenic and peritoneal cavity precursors. Taken together, these findings demonstrate that osteoclast precursors are not identical in the tissues examined. To address the question of why mature osteoclasts occur only in association with bone, we may characterize not only the microenvironment for osteoclastogenesis, but also the osteoclast precursor itself in intramedullary and extramedullary tissues.

Animals↗
PubMed2003

Discrete types of osteoclast precursors can be generated from embryonic stem cells.

Osteoclast precursors (OCPs) share some characteristics with the monocyte/macrophage lineages, but the early events of OCP development are not yet clear. To investigate osteoclastogenesis from the earliest stage, we used step-wise cultures of embryonic stem (ES) cells to induce mature osteoclasts and assessed the effect of vascular endothelial growth factor receptor (VEGFR)-1/Fc chimeric protein on osteoclast development. Addition of VEGFR-1/Fc for the first 5 days of culture (phase I) severely inhibited the development of OCPs. Although OCPs were detected after culturing for a further 5 days (phase II), the reduction of OCPs in phase I was maintained in phase II. The generation of OCPs in phase I was resistant to signal blocking mediated by Kit receptors, but that in phase II was partially inhibited by either an anti-Kit antagonistic antibody or VEGFR-1/Fc and was severely inhibited by the combination of both reagents. Moreover, the OCPs in phase I gave rise to larger numbers of osteoclasts but required a longer period for maturation than the OCPs in phase II. We thus showed that OCPs expanded in phase II, but the majority of OCPs arose from ES cells in a manner dependent on VEGFR-1 binding factor(s) in phase I.

Animals↗
PubMed2002

Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.

Osteoclasts are derived from hematopoietic precursor cells belonging to the monocyte/macrophage lineage. Osteoclast development has been reported to be regulated by several molecules such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor (NF)-kappaB ligand (RANKL), and a decoy receptor of RANKL, osteoprotegerin (OPG). Recently, it was demonstrated that the Notch signaling pathway regulates myeloid differentiation and antagonizes cell fate determination, however, the effect of Notch signaling on the osteoclast lineage has not been reported. In this study, we examined the effect of signaling via Notch receptors on the differentiation into osteoclasts by using cells from the bone marrow, spleen, and peritoneal cavity, and a cloned macrophagelike cell line. Osteoclastogenesis was inhibited by an immobilized Notch ligand, Delta-1. The dish-adherent bone marrow cells precultured with M-CSF expressed both Mac-1 and M-CSF receptors, c-Fms; osteoclastogenesis of these cells was efficiently inhibited. The immobilized Delta-1 also down-regulated the surface c-Fms expression, while the c-Fms gene expression was not changed. Genes for Notch receptors and Notch ligands are expressed in not only hematopoietic cells but also stromal cells that support osteoclast development. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF gene expression, resulting in reduction of their ability to support osteoclast development. Taken together, these findings indicate that Notch signaling affects both osteoclast precursors and stromal cells and thereby negatively regulates osteoclastogenesis.

Animals↗