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Satomi Nishikawa

Publications and source records attributed to Satomi Nishikawa.

At least 19 recordsLinked to original sources
PubMed2006

B cell potential can be obtained from pre-circulatory yolk sac, but with low frequency.

The ontogenic source of definitive hematopoietic system has been identified in non-mammalian vertebrates such as birds and amphibians by orthotopic embryo grafting, but remains unclear for mammals because of technical difficulties. Here, we successfully generated mouse chimeras by grafting yolk sac (YS) on YS of the host embryos before establishing circulation between YS and embryo proper and cultured the whole embryo for 66 h. Donor YS were isolated from C57BL/6 Ly-5.1 and EGFP-transgenic mouse embryos, and recipient embryos from C57BL/6 Ly-5.2 mouse. Almost one-half of the grafts in YS-YS chimeras survived and had obvious blood flow; graft-derived cells comprised 12.7+/-0.9% of the blood cells in the circulation. These graft-derived blood cells consisted mainly of erythroid cells, some myeloid cells and a few blastic cells. In addition, CD19(+) B cells were generated from the graft-derived cells isolated from aorta-gonad-mesonephros (AGM) regions of the YS-YS chimeras; however, the frequency of the YS-derived B cell was low (1.0+/-0.6%) when co-cultured with OP9 stromal cells. These results demonstrate that B cell potential exists in YS before the circulation. Although the major source for B cell is intra-embryonic AGM region, YS may contribute to definitive lymphopoiesis in vivo in mice.

Animals↗
PubMed2006

Platelet derived growth factor receptor alpha is essential for establishing a microenvironment that supports definitive erythropoiesis.

The hematopoietic system undergoes a qualitative change during the embryogenesis of most vertebrates. It is designated as the shift of primitive to definitive hematopoiesis and suitable microenvironment must be established to support this shift. While studying the role of platelet derived growth factor receptor alpha (PDGFR alpha) in embryonic hematopoiesis, we found that it was expressed in a stromal cell component of liver, a major site of this shift, but not in the yolk sac, the site of primitive hematopoiesis. Thus, we considered that development of PDGFRalpha positive stromal cells is an essential requirement for this shift. Without PDFGRalpha positive cell component, erythropoiesis was suppressed in the culture of fetal liver. Moreover, injection of an antagonistic anti-PDGFRalpha monoclonal antibody during embryogenesis suppressed the production of definitive erythrocytes. These indicated that PDGF exerts its effect on a subset of stromal components to prepare a microenvironment that can support the definitive erythropoiesis.

Animals↗
PubMed2006

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development.

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.

Cell Differentiation↗
PubMed2006

Histopathological characteristics of Ito cells and Kupffer cells in the feline liver.

The histopathological characteristics of Ito cells and Kupffer cells were investigated in the liver of 21 cats (age range: 6 months -18 years) autopsied in our laboratory during 2003. Immunohistochemical examinations were performed using antibodies against lysozyme, desmin and alpha-smooth muscle actin. No Kupffer cells reacted with the antibody against lysozyme. However, macrophages in the lung and spleen showed a positive reaction with the antibody. This finding suggests a possibility that the amount of lysozyme in the Kupffer cells of feline liver is comparatively small. On the other hand, large vacuole-laden cells were observed in the hepatic perisinusoid of some feline cases, and these cells showed a positive reaction with antibodies against desmin and alpha-smooth muscle actin. These cells could be Ito cells with large lipid vacuoles. This conclusion was supported by electron microscopic observation and oil red O staining. However, no such large vacuole-laden perisinusoidal cells were detected in the liver of young cats less than 2 years old. The present study revealed the histopathological features of Kupffer cells and Ito cells in the feline liver.

Actins↗
PubMed2005

In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility.

Endothelial cells (ECs) are thought to be derived mainly from the vascular endothelial growth factor receptor 2 (VEGFR-2)+ lateral mesoderm during early embryogenesis. In this study, we specified several pathways for EC differentiation using a murine embryonic stem (ES) cell differentiation culture system that is a model for cellular processes during early embryogenesis. Based on the results of in vitro fate analysis, we show that, in the main pathway, committed ECs are differentiated through the VEGFR-2+ platelet-derived growth factor receptor alpha (PDGFR-alpha)- single-positive (VSP) population that is derived from the VEGFR-2+ PDGFR-alpha+ double-positive (DP) population. This major differentiation course was also confirmed using DNA microarray analysis. In addition to this main pathway, however, ECs also can be generated from the VEGFR-2- PDGFR-alpha+ single-positive (PSP) population, which represents the paraxial mesodermal lineage and is also derived from the DP population. Our results strongly suggest that, even after differentiation from the common progenitor DP population into the VSP and PSP populations, these two populations continue spontaneous switching of their surface phenotype, which results in switching of their eventual fates. The rate of this interlineage conversion between VSP and PSP is unexpectedly high. Because of this potential to undergo fate switch, we conclude that ECs can be generated via multiple pathways in in vitro ES cell differentiation.

Animals↗
PubMed2005

Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells.

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.

Animals↗
PubMed2005

Characterization of mesendoderm: a diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture.

Bipotent mesendoderm that can give rise to both endoderm and mesoderm is an established entity from C. elegans to zebrafish. Although previous studies in mouse embryo indicated the presence of bi-potent mesendoderm cells in the organizer region, characterization of mesendoderm and its differentiation processes are still unclear. As bi-potent mesendoderm is implicated as the major precursor of definitive endoderm, its identification is also essential for exploring the differentiation of definitive endoderm. In this study, we have established embryonic stem (ES) cell lines that carry GFP gene in the goosecoid (Gsc) gene locus and have investigated the differentiation course of mesendodermal cells using Gsc expression as a marker. Our results show that mesendoderm is represented as a Gsc-GFP+ E-cadherin(ECD)+ PDGFRalpha(alphaR)+ population and is selectively induced from ES cells under defined conditions containing either activin or nodal. Subsequently, it diverges to Gsc+ ECD+ alphaR- and Gsc+ ECD- alphaR+ intermediates that eventually differentiate into definitive endoderm and mesodermal lineages, respectively. The presence of mesendodermal cells in nascent Gsc+ ECD+ alphaR+ population was also confirmed by single cell analysis. Finally, we show that the defined culture condition and surface markers developed in this study are applicable for obtaining pure mesendodermal cells and their immediate progenies from genetically unmanipulated ES cells.

Activins↗
PubMed2005

Involvement of Runx1 in the down-regulation of fetal liver kinase-1 expression during transition of endothelial cells to hematopoietic cells.

During early mouse embryogenesis, fetal liver kinase-1 (Flk-1), a receptor for vascular endothelial growth factor, and Runx1, a runt domain transcription factor, have prerequisite roles in the generation of hematopoietic lineages. Flk-1 expression is maintained in successive stages from mesodermal to endothelial cells and is down-regulated in nascent hematopoietic cells, whereas Runx1 (Runt-related transcription factor 1) is expressed in embryonic sites of hematopoietic cell de novo generation and in practically all hematopoietic organs. Here we show that Runx1 represses Flk-1 during the development of hemogenic endothelial cells into hematopoietic cells. We established embryonic stem cell clones carrying the Venus gene, a modified version of yellow fluorescence protein, in the Runx1 locus and cultured them on OP9 cells. Flk-1+ cells appeared on day 3.5, and Runx1+ cells first appeared from the Flk-1+ fraction on day 4.5. The Flk-1+Runx1+ cells rapidly stopped expressing Flk-1 with further incubation and eventually gave rise to CD45+ or TER119+ cells. Runx1 repressed Flk-1 promoter transcriptional activity in an endothelial cell line, and this repression required intact DNA-binding and transactivating domains of Runx1 protein. The repressor activity of Runx1 endogenous Flk-1 was also confirmed overexpressing Runx1 in embryonic stem cell differentiation cultures. These results provide novel insight into the role Runx1 during the development of hematopoietic cell lineages.

Animals↗
PubMed2005

High level of endothelial cell-specific gene expression by a combination of the 5' flanking region and the 5' half of the first intron of the VE-cadherin gene.

To develop a tool to obtain a high level of gene expression specifically in endothelial cells (ECs), we assessed enhancer activity of fragments in the first intron of the VE-cadherin gene using 3 different experimental systems: luciferase assay in the F2 EC line, green fluorescent protein (GFP) expression in ECs generated in embryonic stem (ES) cell differentiation culture, and GFP expression in transgenic mice. Although the 2.5-kbp (kilobase pair) 5' flanking sequence of the VE-cadherin gene is EC specific, adding 4 kbp of the 5' half of the first intron affected an enhancement of the gene expression level in all 3 assay systems. No other fragments tested in this study could confer such effects. Compared with other gene expression units, the unit described in this study would be the most optimum one available to date for EC-specific gene expression. Because this unit can express genes in VE-cadherin(+) progenitors of hematopoietic cells but not in fully committed hematopoietic cells, it will be useful to manipulate specifically the uncommitted progenitor stage during hematopoietic cell differentiation.

Animals↗
PubMed2004

Neural crest and the origin of ectomesenchyme: neural fold heterogeneity suggests an alternative hypothesis.

The striking similarity between mesodermally derived fibroblasts and ectomesenchyme cells, which are thought to be derivatives of the neural crest, has long been a source of interest and controversy. In mice, the gene encoding the alpha subunit of the platelet-derived growth factor receptor (PDGFRalpha) is expressed both by mesodermally derived mesenchymal cells and by ectomesenchyme. Whole-mount immunostaining previously revealed that PDGFRalpha is present in the cephalic neural fold epithelium of early murine embryos (Takakura et al. [1997] J Histochem Cytochem 45:883-893). We now show that, within the neural fold, a sharp boundary exists between E-cadherin-expressing non-neural epithelium and the neural epithelium of the dorsal ridge. In addition, we found that cells coexpressing E-cadherin and PDGFRalpha are present in the non-neural epithelium of the neural folds. These observations raise the possibility that at least some PDGFRalpha(+) ectomesenchyme originates from the lateral non-neural domain of neural fold epithelium. This inference is consistent with previous reports (Nichols [ 1981] J Embryol Exp Morphol 64:105-120; Nichols [ 1986] Am J Anat 176:221-231) that mesenchymal cells emerge precociously from an epithelial neural fold domain resembling the primitive streak in the early embryonic epiblast. Therefore, we propose the name "metablast" for this non-neural epithelial domain to indicate that it is the site of a delayed local delamination of mesenchyme similar to involution of mesoderm during gastrulation. We further propose the testable hypothesis that neural crest and ectomesenchyme are developmentally distinct progenitor populations and that at least some ectomesenchyme is metablast-derived rather than neural crest-derived tissue. Developmental Dynamics 229:118-130, 2004.

Animals↗
PubMed2003

Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis.

The transcription factor recombination signal sequence-binding protein Jkappa (RBP-J) is a key downstream element in the signaling pathway of all four mammalian Notch receptors that are critically involved in the control of embryonic and adult development. RBP-J-deficient mice display complex defects and die around day 9.5 postcoitum. Here, we investigate the function of RBP-J in the development of mesodermal cell lineages by using the OP9 stroma coculture system. RBP-J-deficient embryonic stem (ES) cells gave rise to cardiomyocytes, endothelial cells, and primitive and definitive hematopoietic cells. Thus, RBP-J-mediated signals are not required for generation of these cell types. However, when compared with parental RBP-J-expressing ES cells, cardiomyogenesis derived from RBP-J-deficient ES cells was increased. Repression over the cardiogenic pathway was restored by expressing RBP-J in RBP-J-deficient ES cells. Our data indicate that Notch signaling via RBP-J plays an important role for the correct specification of myocardial cell fates.

Animals↗
PubMed2003

Putative intermediate precursor between hematogenic endothelial cells and blood cells in the developing embryo.

During embryogenesis, endothelial cells are a source of hematopoietic cells. Vascular endothelial (VE)-cadherin modulates adherens junctions between endothelial cells. How endothelial cells, integrated into the vascular bed via adherens junctions, give rise to free-floating hematopoietic cells has been examined. Contrary to our previous reports, in this report a cell type simultaneously expressing VE-cadherin and the hematopoietic marker CD45 was identified, without rigorous enzymatic dissociation of embryonic tissues. In spite of expressing several other endothelial markers such as endothelial cell nitrous oxide synthase (ECNOS) and MECA-32, this newly defined population failed to produce endothelial colonies when cultured on OP9 stroma, in direct contrast to enzymatically dissociated VE-cadherin+ cells. When isolated from 9.5 days post coitus (d.p.c.) embryos, VE-cadherin+ CD45+ cells generated erythroid, myeloid, but not B lymphoid, cells, also in contrast to VE-cadherin+ cells obtained by enzymatic dissociation. Runx1 null mutant embryos lacked this novel population. Collectively, these results introduce a novel VE-cadherin+ population within the developing embryo, which may represent an intermediate cell type in the transition of hemogenic endothelial cells into blood.

Animals↗
PubMed2002

Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage.

We demonstrated that Flk-1(+) cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1(+) LacZ-expressing undifferentiated VPCs. Additional culture of these VPCs with vascular endothelial growth factor (VEGF) resulted in a mixture of ECs and MCs (differentiated VPCs). We injected VPCs subcutaneously into tumor-bearing mice. Five days after the injection, whereas undifferentiated VPCs were often detected as nonvascular cells, differentiated VPCs were more specifically incorporated into developing vasculature mainly as ECs. VPC-derived MCs were also detected in vascular walls. Furthermore, transplantation of differentiated VPCs augmented tumor blood flow in nude mice. These results indicate that a specific vascular contribution in adult neovascularization can be achieved by selective transplantation of ES cell-derived VPCs in appropriate differentiation stages, which should be the basis for vascular regeneration schemes.

Animals↗
PubMed2002

A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells.

Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin. VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF. These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.

Animals↗
PubMed2002

Modulation of VEGFR-2-mediated endothelial-cell activity by VEGF-C/VEGFR-3.

Vascular endothelial growth factor (VEGF) receptor 3 (VEGFR-3), a receptor for VEGF-C, was shown to be essential for angiogenesis as well as for lymphangiogenesis. Targeted disruption of the VEGFR-3 gene in mice and our previous study using an antagonistic monoclonal antibody (MoAb) for VEGFR-3 suggested that VEGF-C/VEGFR-3 signals might be involved in the maintenance of vascular integrity. In this study we used an in vitro embryonic stem (ES) cell culture system to maintain the VEGFR-3(+) endothelial cell (EC) and investigated the role of VEGFR-3 signals at the cellular level. In this system packed clusters of ECs were formed. Whereas addition of exogenous VEGF-A induced EC dispersion, VEGF-C, which can also stimulate VEGFR-2, promoted EC growth without disturbing the EC clusters. Moreover, addition of AFL4, an antagonistic MoAb for VEGFR-3, resulted in EC dispersion. Cytological analysis showed that VEGF-A- and AFL4-treated ECs were indistinguishable in many aspects but were distinct from the cytological profile induced by antagonistic MoAb for VE-cadherin (VECD-1). As AFL4- induced EC dispersion requires VEGF-A stimulation, it is likely that VEGFR-3 signals negatively modulate VEGFR-2. This result provides new insights into the involvement of VEGFR-3 signals in the maintenance of vascular integrity through modulation of VEGFR-2 signals. Moreover, our findings suggest that the mechanisms underlying AFL4-induced EC dispersion are distinct from those underlying VECD-1-induced dispersion for maintenance of EC integrity.

Animals↗
PubMed2002

Blockade of platelet-derived growth factor receptor-beta pathway induces apoptosis of vascular endothelial cells and disrupts glomerular capillary formation in neonatal mice.

Platelet-derived growth factor (PDGF), a potent chemotactic and proliferation factor for mesenchymal-derived cells, has been demonstrated to play critical roles in kidney development. Two receptors for PDGF, PDGFR-alpha and PDGFR-beta, have been identified and we previously analyzed the effects of blockade of PDGFR-alpha signal in neonatal mice. In the current study, we examined the role of PDGFR-beta in glomerular development by blocking PDGFR-beta signal in neonatal mice by administration of antagonistic anti-PDGFR-beta monoclonal antibody. Unlike the mice injected with anti-PDGFR-alpha antibody, the mice injected daily with anti-PDGFR-beta antibody could be kept alive at least for 2 weeks after birth but showed severe disruption of the glomerular structure, whereas no apparent deformation was observed in the collecting ducts. In the disrupted glomeruli, the number of the mesangial cells was reduced markedly. Electron microscopic analysis and immunohistochemical studies with terminal deoxynucleotidyl transferase nick-end labeling staining revealed that the capillary endothelial cells of the glomeruli in the outer cortex region underwent apoptosis. However, the glomeruli located near the medulla were less affected. Because PDGFR-beta is not expressed in the endothelial cells, the effects of the blockade of PDGFR-beta might have caused glomerular endothelial cell apoptosis by inducing the loss of mesangial cells and/or pericytes.

Animals↗
PubMed2002

Definitive hematopoietic commitment within the embryonic vascular endothelial-cadherin(+) population.

OBJECTIVE: The aim of this study was to assess the potential of FLK1(+) and vascular endothelial (VE)-cadherin(+) populations from different stages of embryonic development to generate hematopoietic cells ex vivo and to contribute to the hematopoietic systems of recipient mice. MATERIALS AND METHODS: FLK1(+) of VE-cadherin(+) cells were isolated from 7.5- to 9.5-dpc concepti and cultured ex vivo on OP9 stromal cells and hematopoietic development examined. VE-cadherin(+)CD45(-) cells from 8.5- and 9.5-dpc concepti were injected intrahepatically into newborn busulfan-treated SCID recipients and engraftment monitored. RESULTS: VE-cadherin(+) cells from 7.5- and 8.5-dpc concepti can readily generate hematopoi-etic cells ex vivo compared to FLK1(+) VE-cadherin(-) cells. Similar hematopoietic potential can be found in the VE-cadherin(+) cells from the 8.5-dpc yolk sac. When VE-cadherin(+)CD45(-) cells were injected into SCID recipients, long-term engraftment, particularly within the lymphoid system, was observed. This potential was observed in VE-cadherin(+)CD45(-) cells from 9.5-dpc embryo or yolk sac but from tissues from younger concepti. CONCLUSIONS: FLK1(+)VE-cadherin(-) cells, possibly representing the lateral plate mesoderm, are not as effective at generating hematopoietic cells compared to similarly staged VE-cadherin(+) cells. VE-cadherin(+)CD45(-) cells can also contribute to the hematolymphoid system of intrahepatically injected newborn SCID recipients, suggesting that cells bearing an endothelial phenotype are capable of generating long-term hematopoietic precursors.

Animals↗