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

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2675-2678 ◽  
Author(s):  
Takami Yurugi-Kobayashi ◽  
Hiroshi Itoh ◽  
Jun Yamashita ◽  
Kenichi Yamahara ◽  
Hideyo Hirai ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Mural Cells ◽  
Vascular Walls ◽  
Vascular Regeneration ◽  
Vascular Progenitor Cells ◽  
Differentiation Stage ◽  
Endothelial Growth Factor

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.

Roles of vascular endothelial growth factor receptor 3 signaling in differentiation of mouse embryonic stem cell–derived vascular progenitor cells into endothelial cells

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2372-2379 ◽  
Author(s):  
Hiroyuki Suzuki ◽  
Tetsuro Watabe ◽  
Mitsuyasu Kato ◽  
Keiji Miyazawa ◽  
Kohei Miyazono
Keyword(s):  
Endothelial Cells ◽  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Endothelial Differentiation ◽  
Mouse Es Cells ◽  
Vascular Progenitor Cells ◽  
Vegfr2 Signaling ◽  
Endothelial Growth Factor

AbstractVascular endothelial growth factor receptor 2 (VEGFR2/Flk-1)–positive cells derived from embryonic stem (ES) cells serve as vascular progenitors, which differentiate into endothelial cells (ECs) in the presence of VEGF-A. VEGFR3/Flt-4 (fms-like tyrosine kinase 4) signaling is known to be important for the development of lymphatic endothelial cells (LECs). To elucidate the roles of VEGFR3 signaling in the differentiation of vascular progenitor cells into ECs, we introduced various types of VEGFR3 cDNAs into mouse ES cells. VEGF-C, a ligand for VEGFR2 and VEGFR3, stimulated the endothelial differentiation of the VEGFR2+ cells transfected with the VEGFR3 cDNA but not those transfected with kinasenegative mutants of VEGFR3. The VEGFR3-transfected ECs exhibited high expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), one of the markers of LECs, and showed efficient binding of hyaluronan. VEGF-C(C152S), which is able to activate VEGFR3 but not VEGFR2, failed to induce the endothelial differentiation of mock- and VEGFR3-transfected VEGFR2+ cells, suggesting the essential role of VEGFR2 signaling for endothelial differentiation. Furthermore, kinase-negative mutants of VEGFR3 prevented the VEGF-C–mediated endothelial differentiation of the vascular progenitor cells. Thus, VEGFR2 signaling is required for the endothelial differentiation of mouse ES cells induced by VEGF-C, and VEGFR3 signaling may confer lymphatic endothelial-like phenotypes to ECs.

Transplantation of Motoneuron-Enriched Neural Cells Derived from Mouse Embryonic Stem Cells Improves Motor Function of Hemiplegic Mice

2003 ◽  
Vol 12 (5) ◽  
pp. 457-468 ◽  
Author(s):  
Shunmei Chiba ◽  
Yasumasa Iwasaki ◽  
Hiroaki Sekino ◽  
Noboru Suzuki
Keyword(s):  
Motor Cortex ◽  
Progenitor Cells ◽  
Neural Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Progenitor ◽  
Cortical Region ◽  
Neural Cells ◽  
Es Cell ◽  
Neuroprotective Effects

Embryonic stem (ES) cells are expected to be a potential donor source for neural transplantation. We have obtained motoneuron-enriched neural progenitor cells by culturing mouse ES cells with retinoic acid (RA). The cells also expressed mRNA of a neurotrophic factor, neurotrophin-3 (NT-3). The left motor cortex area of mice was damaged by cryogenic brain injury, and the neural cells were transplanted underneath the injured motor cortex, neighboring to the paraventricular region. We found that the cells expressing neuronal phenotypes not only remained close to the implantation site, but also exhibited substantial migration penetrating into the damaged lesion, in a seemingly directed manner up to cortical region. We found that some of the neural cells differentiated into Islet1-positive motoneurons. It seems likely that the ability of the ES cell-derived neural progenitor cells to respond in vivo to guidance cues and signals that can direct their migration and differentiation may contribute to functional recovery of the recipient mice. We found that an “island of the mature neuronal cells” of recipient origin emerged in the damaged motor cortex. This may be associated with the neuroprotective effects of the ES cell-derived neural cells. The ES cells differentiated into CD31+ vasculoendothelial cells with the RA treatment in vitro. Furthermore, the grafted cells may provide sufficient neurotrophic factors such as NT-3 for neuroprotection and regeneration. The grafted neural cells that migrated into residual cortex and differentiated into neurons had purposefully elongated axons that were stained with anti-neurofilament middle chain (NFM) antibody. Our study suggests that motoneurons can be induced from ES cells, and ES cells become virtually an unlimited source of cells for experimental and clinical neural cell transplantation.

Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 912-920 ◽  
Author(s):  
Jody J. Haigh ◽  
Masatsugu Ema ◽  
Katharina Haigh ◽  
Marina Gertsenstein ◽  
Peter Greer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Es Cells ◽  
Consensus Sequence ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Developmental Potential ◽  
Vascular Progenitor Cells ◽  
Endothelial Growth Factor

AbstractRelatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A–mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A–independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell–derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

scholarly journals Ras signaling directs endothelial specification of VEGFR2+ vascular progenitor cells

2008 ◽  
Vol 181 (1) ◽  
pp. 131-141 ◽  
Author(s):  
Kyoko Kawasaki ◽  
Tetsuro Watabe ◽  
Hitoshi Sase ◽  
Masanori Hirashima ◽  
Hiroshi Koide ◽  
...  
Keyword(s):  
Growth Factor ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Ras Signaling ◽  
Whole Embryo Culture ◽  
Extracellular Signal ◽  
Vascular Progenitor Cells ◽  
Endothelial Growth Factor

Vascular endothelial growth factor receptor 2 (VEGFR2) transmits signals of crucial importance to vasculogenesis, including proliferation, migration, and differentiation of vascular progenitor cells. Embryonic stem cell–derived VEGFR2+ mesodermal cells differentiate into mural lineage in the presence of platelet derived growth factor (PDGF)–BB or serum but into endothelial lineage in response to VEGF-A. We found that inhibition of H-Ras function by a farnesyltransferase inhibitor or a knockdown technique results in selective suppression of VEGF-A–induced endothelial specification. Experiments with ex vivo whole-embryo culture as well as analysis of H-ras−/− mice also supported this conclusion. Furthermore, expression of a constitutively active H-Ras[G12V] in VEGFR2+ progenitor cells resulted in endothelial differentiation through the extracellular signal-related kinase (Erk) pathway. Both VEGF-A and PDGF-BB activated Ras in VEGFR2+ progenitor cells 5 min after treatment. However, VEGF-A, but not PDGF-BB, activated Ras 6–9 h after treatment, preceding the induction of endothelial markers. VEGF-A thus activates temporally distinct Ras–Erk signaling to direct endothelial specification of VEGFR2+ vascular progenitor cells.

SDF-1/CXCL12 Enhances Survival of Murine Embryonic Stem Cells and Their Production of Myeloid Progenitors, but Not Hemangioblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1693-1693
Author(s):  
Ying Guo ◽  
Giao Hangoc ◽  
Huimin Bian ◽  
Louis M. Pelus ◽  
Hal E. Broxmeyer
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Receptor Expression ◽  
Hematopoietic Progenitor Cells ◽  
Es Cell ◽  
Hematopoietic Progenitor ◽  
Myeloid Progenitors

Abstract Stromal cell derived factor (SDF)-1/CXCL12 has been shown to promote the survival of embryonic retinal ganglion cells, peritoneal B1a (PerBla) lymphocytes and chronic lymphocytic leukemia B cells. Our previous studies implicated SDF-1 as an important factor in enhancing survival of murine bone marrow (BM) hematopoietic stem cells and, human cord blood and adult human BM myeloid progenitors. Since pluripotent embryonic stem (ES) cells can give rise to differentiated cell types derived from all three primary germ layers (endoderm, mesoderm, and ectoderm) and adult stem cells are generated during embryoid body (EB) formation, we investigated whether SDF-1 has effects on survival of ES cells and EB generation of hematopoietic progenitor cells. In order to establish SDF-1 expression patterns during EB formation, we screened supernatants during day 1–5 EB formation for SDF-1 production by three murine ES cell lines (E14, R1 and CCE). We observed low but detectable SDF-1 secreted in cultures of ES cells and day 1 stage EBs. SDF-1 was increased in the media from day 2 stage EBs and continued to increase through days 4–5. CXCR4, SDF-1 receptor, expression was also analyzed. CXCR4 mRNA expression was low in ES cells and day 1–3 EBs, and increased significantly from Day 4 EBs, reaching maximum levels at day 5, and decreasing after day 6. Surface CXCR4 expression was consistent with mRNA data. To determine if SDF-1 had an effect on ES cell survival, we cultured ES cells without serum, and added serum at either 0, 24, 48 or 96 hrs to each of the following groups: A) Control, B) SDF-1 (100ng/ml) or C) AMD3100 (1 μM), an SDF-1 receptor (CXCR4) antagonist. Colonies were scored 7 days after the addition of serum. SDF-1 enhanced survival of ES cells, while AMD3100 decreased survival. We also checked the apoptosis of ES cells after withdrawing serum for 24, 48, 72 and 96 hours in four groups: A) control, B) SDF-1 (100ng/ml), C) AMD3100 (1 μM) or D) AMD3100 (1 μM) and SDF-1 (100ng/ml). SDF-1 decreased apoptosis and AMD3100 blocked the SDF-1 effect. AMD3100 alone increased apoptosis compared to control. This suggests that AMD3100 blocked endogenous SDF-1 effects. To determine if SDF-1 had an effect on differentiation of hematopoietic progenitor cells, we added SDF-1 (100 ng/ml), AMD3100, or AMD3100 plus SDF-1 (100 ng/ml) at the beginning of EB formation, immediately after removal of LIF, and quantitated primitive erythroid (p-BFU-E), definitive erythroid (d-BFU-E), granulocyte-macrophage (CFU-GM) and multipotential Granulocyte/Erythroid/Macrophage/Megakaryocyte (CFU-GEMM) colony formation. In comparison to control cells (cultured without SDF-1 and AMD3100), SDF-1 increased numbers of p-BFU-E, d-BFU-E, CFU-GM, and CFU-GEMM colonies. Addition of AMD3100 with SDF-1 blocked the enhancing effect of SDF-1. In addition, significantly decreased numbers of colonies were also observed in the presence of AMD3100 alone. This suggests that AMD3100 blocks endogenous SDF-1 actions, consistent with our data on SDF-1 production during EB formation. In order to determine when SDF-1 starts affecting hematopoiesis, hemangioblast colony assays were used. Neither SDF-1 nor AMD3100 influenced hemangioblast colony formation or expression of Flk-1 mRNA, a marker of hemangioblasts. The results suggest a role for SDF-1 in ES cell growth and differentiation.

scholarly journals Production of Mice Entirely Derived from Embryonic Stem (ES) Cell with Many Passages by Coculture of ES Cells with Cytochalasin B Induced Tetraploid Embryos.

1995 ◽  
Vol 44 (3) ◽  
pp. 205-210 ◽  
Author(s):  
Otoya UEDA ◽  
Kouichi JISHAGE ◽  
Nobuo KAMADA ◽  
Satomi UCHIDA ◽  
Hiroshi SUZUKI
Keyword(s):  
Cytochalasin B ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

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