Hidetoshi Yamazaki

Derivation of melanocytes from embryonic stem cells in culture

We report that embryonic stem (ES) cells were efficiently induced to differentiate to melanocytes in vitro. When undifferentiated ES cells were cocultured with a bone marrow–derived stromal cell line, a very small but significant number of melanocytes were reproducibly generated. This process was greatly enhanced by addition of dexamethasone to the culture and strictly depended on steel factor, the ligand for the c‐Kit receptor tyrosine kinase. Expression of c‐Kit on the precursor cells was confirmed by using SCL/tal‐1‐/‐ ES cells, which are defective for producing hematopoietic cells, which were thus ruled out as possible sources of nonmelanogenic c‐Kit‐expressing cells. The morphology, reactivity to growth factors, and expression of melanogenic markers of the cells generated all indicated unequivocally that these cells were melanocytes. This culture system may provide a potent tool for the study of development and function of melanocytes. Dev Dyn 1999;216:450–458. ©1999 Wiley‐Liss, Inc.

Generation of Structures Formed by Lens and Retinal Cells Differentiating From Embryonic Stem Cells

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye‐like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye‐like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye. Developmental Dynamics 228:664–671, 2003.

Retinoic acid receptor α dominant negative form causes steatohepatitis and liver tumors in transgenic mice

Although attention has focused on the chemopreventive action of retinoic acid (RA) in hepatocarcinogenesis, the functional role of RA in the liver has yet to be clarified. To explore the role of RA in the liver, we developed transgenic mice expressing RA receptor (RAR) α– dominant negative form in hepatocytes using albumin promoter and enhancer. At 4 months of age, the RAR α– dominant negative form transgenic mice developed microvesicular steatosis and spotty focal necrosis. Mitochondrial β‐oxidation activity of fatty acids and expression of its related enzymes, including VLCAD, LCAD, and HCD, were down‐regulated; on the other hand, peroxisomal β‐oxidation and its related enzymes, including AOX and BFE, were up‐regulated. Expression of cytochrome p4504a10, cytochrome p4504a12, and cytochrome p4504a14 was increased, suggesting that ω‐oxidation of fatty acids in microsomes was accelerated. In addition, formation of H2O2 and 8‐hydroxy‐2′‐deoxyguanosine was increased. After 12 months of age, these mice developed hepatocellular carcinoma and adenoma of the liver. The incidence of tumor formation increased with age. Expression of β‐catenin and cyclin D1 was enhanced and the TCF‐4/β‐catenin complex was increased, whereas the RAR α/ β‐catenin complex was decreased. Feeding on a high‐RA diet reversed histological and biochemical abnormalities and inhibited the occurrence of liver tumors. These results suggest that hepatic loss of RA function leads to the development of steatohepatitis and liver tumors. In conclusion, RA plays an important role in preventing hepatocarcinogenesis in association with fatty acid metabolism and Wnt signaling. (HEPATOLOGY 2004;40:366–375.)

Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis.

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.

Wnt Signaling Regulates Hemopoiesis Through Stromal Cells

Hemopoietic cells develop in a complex milieu that is made up of diverse components, including stromal cells. Wnt genes, which are known to regulate the fate of the cells in a variety of tissues, are expressed in hemopoietic organs. However, their roles in hemopoiesis are not well characterized. In this study, we examined the roles of Wnt proteins in hemopoiesis using conditioned medium containing Wnt-3a. This conditioned medium dramatically reduced the production of B lineage cells and myeloid lineage cells, except for macrophages in the long-term bone marrow cultures grown on stromal cells, although the sensitivity to the conditioned medium differed, depending on the hemopoietic lineage. In contrast, the same conditioned medium did not affect the generation of B lineage or myeloid lineage cells in stromal cell-free conditions. These results suggested that Wnt proteins exert their effects through stromal cells. Indeed, these effects were mimicked by the expression of a stabilized form of β-catenin in stromal cells. In this study, we demonstrated that Wnt signaling regulates hemopoiesis through stromal cells with selectivity and different degrees of the effect, depending on the hemopoietic lineage in the hemopoietic microenvironment.

Distinct Osteoclast Precursors in the Bone Marrow and Extramedullary Organs Characterized by Responsiveness to Toll-Like Receptor Ligands and TNF-α

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-α. 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-α as well as receptor activator of NF-κB ligand in the presence of M-CSF. Splenic osteoclast precursors, even in osteoclast-deficient osteopetrotic mice, differentiated into mature osteoclasts following exposure to TNF-α or receptor activator of NF-κB 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-α. 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.

Osteoclast precursors in bone marrow and peritoneal cavity

Osteoclasts differentiate from cells that share some phenotypes with mature macrophages and monocytes, but early precursors for osteoclasts still remain obscure. To characterize osteoclast precursors, using monoclonal anti‐c‐Fms and anti‐c‐Kit antibodies, bone marrow cells were separated and the frequency of clonogenic progenitors were measured. Osteoclast precursors in the bone marrow mainly expressed c‐Kit and diminished in frequency when they expressed c‐Fms. In contrast to bone marrow, the precursors in the peritoneal cavity were enriched with a population of c‐Fms+. Injection of these antibodies into mice demonstrated that peritoneal osteoclast precursors were sensitive to anti‐c‐Fms but not to anti‐c‐Kit antibodies, whereas those in bone marrow only declined in the presence of both antibodies. Meanwhile, c‐Fms as opposed to c‐Kit played an essential role in the generation of osteoclasts in cultures. We also compared osteoclast precursors with colony forming cells (CFU‐M) by a macrophage colony stimulating factor. CFU‐M in bone marrow decreased when anti‐c‐Kit antibody was administered and no CFU‐M was detected in peritoneum. In this study, we show differences between proliferative potential osteoclast precursors maintained in bone marrow and peritoneum and between CFU‐M and osteoclast precursors. J. Cell. Physiol. 170:241–247, 1997.

Origins and Properties of Dental, Thymic, and Bone Marrow Mesenchymal Cells and Their Stem Cells

Mesenchymal cells arise from the neural crest (NC) or mesoderm. However, it is difficult to distinguish NC-derived cells from mesoderm-derived cells. Using double-transgenic mouse systems encoding P0-Cre, Wnt1-Cre, Mesp1-Cre, and Rosa26EYFP, which enabled us to trace NC-derived or mesoderm-derived cells as YFP-expressing cells, we demonstrated for the first time that both NC-derived (P0- or Wnt1-labeled) and mesoderm-derived (Mesp1-labeled) cells contribute to the development of dental, thymic, and bone marrow (BM) mesenchyme from the fetal stage to the adult stage. Irrespective of the tissues involved, NC-derived and mesoderm-derived cells contributed mainly to perivascular cells and endothelial cells, respectively. Dental and thymic mesenchyme were composed of either NC-derived or mesoderm-derived cells, whereas half of the BM mesenchyme was composed of cells that were not derived from the NC or mesoderm. However, a colony-forming unit-fibroblast (CFU-F) assay indicated that CFU-Fs in the dental pulp, thymus, and BM were composed of NC-derived and mesoderm-derived cells. Secondary CFU-F assays were used to estimate the self-renewal potential, which showed that CFU-Fs in the teeth, thymus, and BM were entirely NC-derived cells, entirely mesoderm-derived cells, and mostly NC-derived cells, respectively. Colony formation was inhibited drastically by the addition of anti-platelet–derived growth factor receptor-β antibody, regardless of the tissue and its origin. Furthermore, dental mesenchyme expressed genes encoding critical hematopoietic factors, such as interleukin-7, stem cell factor, and cysteine-X-cysteine (CXC) chemokine ligand 12, which supports the differentiation of B lymphocytes and osteoclasts. Therefore, the mesenchymal stem cells found in these tissues had different origins, but similar properties in each organ.

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‐β‐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 α 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.

Sequential requirements for SCL/tal-1, GATA-2, macrophage colony-stimulating factor, and osteoclast differentiation factor/osteoprotegerin ligand in osteoclast development

OBJECTIVE Osteoclasts are of hematopoietic origin. The mechanism by which hematopoietic stem cells are specified to the osteoclast lineage is unclear. To understand the process of generation and differentiation of this lineage of cells, we performed in vitro studies on the differentiation of embryonic stem cells. MATERIALS AND METHODS We examined the potential of mutant embryonic stem cell lines harboring targeted deletions of the GATA-1, FOG, SCL/tal-1, or GATA-2 genes to differentiate into osteoclasts and determined when these molecules function in osteoclast development. RESULTS The lack of GATA-1 or FOG did not affect osteoclastogenesis. In contrast, SCL/tal-1-null embryonic stem cells generated no osteoclasts. In the case of the loss of GATA-2, a small number of osteoclasts were generated. GATA-2-null osteoclasts were morphologically normal and the terminal maturation was not disturbed, but a defect was observed in the generation of osteoclast progenitors. Experiments using specific inhibitors that block the signaling through macrophage colony-stimulating factor and osteoclast differentiation factor/osteoprotegerin ligand suggested that GATA-2 seems to act earlier in osteoclastogenesis than these cytokines. Interestingly, macrophage colony-forming units were not severely reduced by the loss of GATA-2 compared to osteoclast progenitors. CONCLUSION These results indicate that osteocalsts need SCL/tal-1 at an early point in development, and that GATA-2 is required for generation of osteoclast progenitors but not for the later stages when macrophage colony-stimulating factor and osteoclast differentiation factor/ osteoprotegerin ligand are needed. We also demonstrated that osteoclast progenitors behave as a different population than macrophage colony-forming units.