scholarly journals The versican-hyaluronan complex provides an essential extracellular matrix niche for Flk1+ hematoendothelial progenitors

2019 ◽  
Author(s):  
Sumeda Nandadasa ◽  
Anna O’Donnell ◽  
Ayako Murao ◽  
Yu Yamaguchi ◽  
Ronald J. Midura ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Mouse Embryos ◽  
Indian Hedgehog ◽  
Vascular Endothelial ◽  
Angiogenic Response ◽  
Cell Lineages ◽  
Primitive Hematopoiesis

AbstractLittle is known about extracellular matrix (ECM) contributions to formation of the earliest cell lineages in the embryo. Here, we show that the proteoglycan versican and glycosaminoglycan hyaluronan are associated with emerging Flk1+ hematoendothelial progenitors at gastrulation. The mouse versican mutant Vcanhdf lacks yolk sac vasculature, with attenuated yolk sac hematopoiesis. CRISPR/Cas9-mediated Vcan inactivation in mouse embryonic stem cells reduced vascular endothelial and hematopoietic differentiation in embryoid bodies, which generated fewer blood colonies, and had an impaired angiogenic response to VEGF165. HA was severely depleted in Vcanhdf embryos, with corresponding increase in the HA-depolymerase TMEM2. Conversely, HA-deficient mouse embryos also had vasculogenic suppression but with increased versican proteolysis. VEGF165 and Indian hedgehog, crucial vasculogenic factors, utilized the versican-HA matrix, specifically versican chondroitin sulfate chains, for binding. Versican-HA ECM is an obligate requirement for vasculogenesis and primitive hematopoiesis, acts as an vasculogenic factor-enriching microniche for Flk1+ progenitors from their origin at gastrulation.

scholarly journals Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3720-3730 ◽  
Author(s):  
M Bielinska ◽  
N Narita ◽  
M Heikinheimo ◽  
SB Porter ◽  
DB Wilson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Cell Stage ◽  
Primitive Hematopoiesis

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

scholarly journals Interaction of retinoic acid and scl controls primitive blood development

Blood ◽  
2010 ◽  
Vol 116 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Jill L. O. de Jong ◽  
Alan J. Davidson ◽  
Yuan Wang ◽  
James Palis ◽  
Praise Opara ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Erythroid Progenitors ◽  
Self Renewal ◽  
Hematopoietic Development ◽  
Blood Island ◽  
Primitive Hematopoiesis

Abstract Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1+ erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4−/− mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

scholarly journals Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 303-316 ◽  
Author(s):  
R. Wang ◽  
R. Clark ◽  
V.L. Bautch
Keyword(s):  
Endothelial Cells ◽  
Blood Vessel ◽  
Light And Electron Microscopy ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Density Lipoprotein ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Vascular Channels

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen ‘flowing’ through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.

scholarly journals Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 860-870 ◽  
Author(s):  
Elias T. Zambidis ◽  
Bruno Peault ◽  
Tea Soon Park ◽  
Fred Bunz ◽  
Curt I. Civin
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Fetal Hemoglobin ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

AbstractWe elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis.

Modulation of hematopoietic and endothelial cell differentiation from mouse embryonic stem cells by different culture conditions

Blood ◽  
2005 ◽  
Vol 105 (1) ◽  
pp. 111-114 ◽  
Author(s):  
Wen Jie Zhang ◽  
Changwon Park ◽  
Elizabeth Arentson ◽  
Kyunghee Choi
Keyword(s):  
Endothelial Cell ◽  
Cell Differentiation ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Hematopoietic Progenitors ◽  
Endothelial Cell Differentiation

Abstract Embryonic stem (ES) cells can differentiate into many different somatic cells in culture. To better correlate hematopoietic and endothelial cell differentiation of ES cells in currently available protocols, we compared fetal liver kinase-1 (Flk-1)–, stem cell leukemia (Scl)–, and vascular endothelial–cadherin (VE-cadherin)–expressing cells generated in embryoid bodies (EBs) and on OP9 cells. We report that the kinetics of Scl and Flk-1 expression were similar in EBs and OP9 cells, although Flk-1 expression was extended on OP9 cells. CD45+ and Ter-119+ cells developed more efficiently in EBs, whereas VE-cadherin+ cells developed largely on OP9 cells. Cell sorting and replating studies showed that Scl+ cells, not Flk-1+ or VE-cadherin+ cells, were enriched for primitive and definitive hematopoietic progenitors. Our studies indicate that optimal hematopoietic and endothelial cell differentiation occur in EBs and on OP9 cells, respectively. Regardless of the culture systems used, Scl is the most relevant marker for enriching primitive and definitive hematopoietic progenitors.

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2003-2013 ◽  
Author(s):  
Maria Teresa Mitjavila-Garcia ◽  
Michel Cailleret ◽  
Isabelle Godin ◽  
Maria Manuela Nogueira ◽  
Karine Cohen-Solal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide

Planta Medica ◽  
2018 ◽  
Vol 84 (11) ◽  
pp. 768-778 ◽  
Author(s):  
Enas Ali ◽  
Fatemeh Sharifpanah ◽  
Maria Wartenberg ◽  
Heinrich Sauer
Keyword(s):  
Nitric Oxide ◽  
Stem Cell ◽  
Protein Expression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Silybum Marianum ◽  
Branching Points ◽  
Vascular Branching

AbstractSilibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.

scholarly journals Neuropilin-1 in regulation of VEGF-induced activation of p38MAPK and endothelial cell organization

Blood ◽  
2008 ◽  
Vol 112 (9) ◽  
pp. 3638-3649 ◽  
Author(s):  
Harukiyo Kawamura ◽  
Xiujuan Li ◽  
Katsutoshi Goishi ◽  
Laurens A. van Meeteren ◽  
Lars Jakobsson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vascular Endothelial ◽  
Loss Of Function ◽  
Sprouting Angiogenesis ◽  
Neuropilin 1 ◽  
Cell Organization ◽  
Vascular Structures ◽  
Endothelial Growth Factor

Abstract Vascular endothelial growth factor (VEGF)–A regulates vascular development and angiogenesis. VEGF isoforms differ in ability to bind coreceptors heparan sulfate (HS) and neuropilin-1 (NRP1). We used VEGF-A165 (which binds HS and NRP1), VEGF-A121 (binds neither HS nor NRP1), and parapoxvirus VEGF-E-NZ2 (binds NRP1 but not HS) to investigate the role of NRP1 in organization of endothelial cells into vascular structures. All 3 ligands induced similar level of VEGFR-2 tyrosine phosphorylation in the presence of NRP1. In contrast, sprouting angiogenesis in differentiating embryonic stem cells (embryoid bodies), formation of branching pericyte-embedded vessels in subcutaneous matrigel plugs, and sprouting of intersegmental vessels in developing zebrafish were induced by VEGF-A165 and VEGF-E-NZ2 but not by VEGF-A121. Analyses of recombinant factors with NRP1-binding gain- and loss-of-function properties supported the conclusion that NRP1 is critical for VEGF-induced sprouting and branching of endothelial cells. Signal transduction antibody arrays implicated NRP1 in VEGF-induced activation of p38MAPK. Inclusion of the p38MAPK inhibitor SB203580 in VEGF-A165–containing matrigel plugs led to attenuated angiogenesis and poor association with pericytes. Our data strongly indicate that the ability of VEGF ligands to bind NRP1 influences p38MAPK activation, and formation of functional, pericyte-associated vessels.

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