Toshiyuki Yamane

Insulin‐Like Growth Factor Promotes Engraftment, Differentiation, and Functional Improvement after Transfer of Embryonic Stem Cells for Myocardial Restoration

Insulin‐like growth factor‐1 (IGF‐1) promotes myocyte proliferation and can reverse cardiac abnormalities when it is administered in the early fetal stage. Supplementation of a mouse embryonic stem cell (ESC) suspension with IGF‐1 might enhance cellular engraftment and host organ‐specific differentiation after injection in the area of acute myocardial injury. In the study reported here, we sought to enhance the restorative effect of ESCs in the injured heart by adding IGF‐1 to the injected cell population. Green fluorescent protein (GFP)–labeled sv129 ESCs (2.5 × 105) were injected into the ischemic area after left anterior descending (LAD) artery ligation in BalbC mice. Recombinant mouse IGF‐1 (25 ng) was added to the cell suspension prior to the injection (n = 5). Echocardiography was performed before organ harvest 2 weeks later. The degree of restoration (ratio of GFP+ to infarct area), expression of cardiac markers by GFP+ cells, inflammatory response, and tumorigenicity were evaluated. Mice with LAD ligation only (n = 5) and ESC transfer without IGF‐1 (n = 5) served as controls. ESCs formed viable grafts and improved cardiac function. Left ventricular wall thickness was higher in the IGF‐1 group (p = .025). There was a trend toward higher fractional shortening in the IGF‐treated group. Histological analysis demonstrated that IGF‐1 promoted expression of α‐sarcomeric actin (p = .015) and major histocompatibility complex class I (p = .01). IGF did not affect the cellular response to the donor cells or tumorigenicity. IGF‐1 promotes expression of cardiomyocyte phenotype in ESCs in vivo. It should be considered as an adjuvant to cell transfer for myocardial restoration.

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.

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.

Bmi-1-Green Fluorescent Protein-Knock-In Mice Reveal the Dynamic Regulation of Bmi-1 Expression in Normal and Leukemic Hematopoietic Cells

The ability to self‐renew is essential for all kinds of stem cells regardless of tissue type. One of the best candidate genes involved in conferring self‐renewal capacity is Bmi‐1, which has been proven to be essential for the maintenance of both normal adult hematopoietic and leukemia stem cells, as well as adult neural stem cells. To investigate the possible role of Bmi‐1 in other cell types that also self‐renew, we generated Bmi‐1‐green fluorescent protein (GFP)‐knock‐in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of the Bmi‐1 gene. Using these targeted reporter mice, we demonstrated that Bmi‐1 is expressed in hematopoietic stem cells (HSCs) at its highest levels and downregulated upon commitment to differentiation. An in vivo reconstitution assay revealed that the frequency of HSCs was 1/16 in Bmi‐1highc‐kit+lin−Sca‐1+ bone marrow (BM) cells and 1/49 in Bmi‐1highlin− BM cells, suggesting that Bmi‐1 may serve as a marker for normal HSCs. In murine leukemia models induced by P210BCR/ABL or TEL/PDGFβR + AML1/ETO, Bmi‐1 was not overexpressed in leukemic HSCs, despite the increase in the HSC numbers. Bmi‐1 was expressed at its highest levels in undifferentiated leukemia cells. Furthermore, in several other nonhematopoietic tissues, cells could be separated into distinct subpopulations with differential Bmi‐1 expression. Thus, these mice allow for the isolation of viable Bmi‐1‐expressing cells and have the potential to become a useful tool for understanding the role of Bmi‐1 in normal and cancer stem cells in multiple tissue types.

Stimulation of Paracrine Pathways With Growth Factors Enhances Embryonic Stem Cell Engraftment and Host-Specific Differentiation in the Heart After Ischemic Myocardial Injury

Background—Growth factors play an essential role in organogenesis. We examine the potential of growth factors to enhance cell engraftment and differentiation and to promote functional improvement after transfer of undifferentiated embryonic stem cells into the injured heart. Methods and Results—Green fluorescent protein (GFP)–positive embryonic stem cells derived from 129sv mice were injected into the ischemic area after left anterior descending artery ligation in allogenic (BALB/c) mice. Fifty nanograms of recombinant mouse vascular endothelial growth factor, fibroblast growth factor (FGF), and transforming growth factor (TGF) was added to the cell suspension. Separate control groups were formed in which only the growth factors were given. Echocardiography was performed 2 weeks later to evaluate heart function (fractional shortening [FS]), end-diastolic diameter, and left ventricular wall thickness). Hearts were harvested for histology (connexin 43, α-sarcomeric actin, CD3, CD11c, major histocompatability complex class I, hematoxylin-eosin). Degree of restoration (GFP-positive graft/infarct area ratio), expression of cardiac markers, host response, and tumorigenicity were evaluated. Cell transfer resulted in improved cardiac function. TGF-β led to better restorative effect and a stronger expression of connexin 43, α-sarcomeric actin, and major histocompatability complex class I. TGF-β and FGF retained left ventricular diameter. FS was better in the TGF-β, FGF, and embryonic stem cells–only group compared with left anterior descending artery–ligated controls. Growth factors with cells (TGF-β, FGF) resulted in higher FS and smaller end-diastolic diameter than growth factors alone. Conclusions—Growth factors can promote in vivo organ-specific differentiation of early embryonic stem cells and improve myocardial function after cell transfer into an area of ischemic lesion. TGF-β should be considered as an adjuvant for myocardial restoration with the use of embryonic stem cells.

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.

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.

Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development.

The development of melanocytes from neural crest‐derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein‐coupled endothelin receptor B (EDNRB) pathways. Loss‐of‐function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21‐day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using KitW‐LacZ/KitW‐LacZ ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of KitW57/KitW57mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3−/− ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings. Developmental Dynamics 233:407–417, 2005.