Specific Effect of 5-Fluorouracil on α-Fetoprotein Gene Expression During the In Vitro Mouse Embryonic Stem Cell Differentiation

2010 ◽  
Vol 29 (3) ◽  
pp. 297-304 ◽  
Author(s):  
David Pamies ◽  
Néstor Vicente-Salar ◽  
Miguel A. Sogorb ◽  
Enrique Roche ◽  
Juan A. Reig
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Screening Methods ◽  
Embryonic Stem Cell Differentiation ◽  
Visceral Endoderm ◽  
Gene Markers ◽  
Wide Range

Embryonic stem (ES) cells are considered an important alternative to develop in vitro screening methods for embryotoxicity. Mouse ES cells can be cultured as cell suspension aggregates termed “embryoid bodies” (EBs) in which cells start to differentiate. We have studied the expression of several genes in the presence of a wide range of concentrations of 5-fluorouracil (5-FU). This well-established embryotoxic compound completely inhibited cell viability at 200 nmol/L in monolayer cultures. At lower concentrations, 5-FU led to decrease in the expression of the α-fetoprotein gene, a marker of the visceral endoderm, in the EBs. However, the expression of several mesodermal gene markers was not significantly affected at these concentrations. These results suggest a high sensitivity of the visceral endoderm differentiation to 5-FU. Therefore, the quantification of the α-fetoprotein gene after exposure to potential embryotoxicants should be considered an additional end point in future embryotoxicity assays in vitro with ES cells.

Filamin A Controls Embryonic Stem Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4599-4599
Author(s):  
Taisuke Kanaji ◽  
Takashi Okamura ◽  
Peter J. Newman
Keyword(s):  
Cell Differentiation ◽  
Signaling Pathways ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Actin Binding ◽  
Embryonic Stem Cell Differentiation ◽  
Filamin A ◽  
Es Cell

Abstract Abstract 4599 Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures: Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Mouse lysocardiolipin acyltransferase controls the development of hematopoietic and endothelial lineages during in vitro embryonic stem-cell differentiation

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3601-3609 ◽  
Author(s):  
Chengyan Wang ◽  
Patrick W. Faloon ◽  
Zhijia Tan ◽  
Yaxin Lv ◽  
Pengbo Zhang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Embryonic Stem Cell Differentiation ◽  
Hematopoietic Stem ◽  
Endothelial Genes

Abstract The blast colony-forming cell (BL-CFC) was identified as an equivalent to the hemangioblast during in vitro embryonic stem (ES) cell differentiation. However, the molecular mechanisms underlying the generation of the BL-CFC remain largely unknown. Here we report the isolation of mouse lysocardiolipin acyltransferase (Lycat) based on homology to zebrafish lycat, a candidate gene for the cloche locus. Mouse Lycat is expressed in hematopoietic organs and is enriched in the Lin−C-Kit+Sca-1+ hematopoietic stem cells in bone marrow and in the Flk1+/hCD4+(Scl+) hemangioblast population in embryoid bodies. The forced Lycat transgene leads to increased messenger RNA expression of hematopoietic and endothelial genes as well as increased blast colonies and their progenies, endothelial and hematopoietic lineages. The Lycat small interfering RNA transgene leads to a decrease expression of hematopoietic and endothelial genes. An unbiased genomewide microarray analysis further substantiates that the forced Lycat transgene specifically up-regulates a set of genes related to hemangioblasts and hematopoietic and endothelial lineages. Therefore, mouse Lycat plays an important role in the early specification of hematopoietic and endothelial cells, probably acting at the level of the hemangioblast.

Na+/H+ exchanger isoform 1 facilitates cardiomyocyte embryonic stem cell differentiation

2009 ◽  
Vol 296 (1) ◽  
pp. H159-H170 ◽  
Author(s):  
Xiuju Li ◽  
Pratap Karki ◽  
Lei Lei ◽  
Huayan Wang ◽  
Larry Fliegel
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Embryonic Stem Cell Differentiation ◽  
Cardiomyocyte Differentiation ◽  
Nhe1 Activity

Embryonic stem cells provide one potential source of cardiomyocytes for cardiac transplantation; however, after differentiation of stem cells in vitro, cardiomyocytes usually account for only a minority of cells present. To gain insights into improving cardiomyocyte development from stem cells, we examined the role of the Na+/H+ exchanger isoform 1 (NHE1) in cardiomyocyte differentiation. NHE1 protein and message levels were induced by treatment of CGR8 cells to form embryoid bodies and cardiomyocytes. The NHE1 protein was present on the cell surface and NHE1 inhibitor-sensitive activity was detected. Inhibition of NHE1 activity during differentiation of CGR8 cells prevented cardiomyocyte differentiation as indicated by decreased message for transcription factors Nkx2-5 and Tbx5 and decreased levels of α-myosin heavy chain protein. Increased expression of NHE1 from an adenoviral vector facilitated cardiomyocyte differentiation. Similar results were found with cardiomyocyte differentiation of P19 embryonal carcinoma cells. CGR8 cells were treated to induce differentiation, but when differentiation was inhibited by dispersing the EBs, myocardial development was inhibited. The results demonstrate that NHE1 activity is important in facilitating stem cell differentiation to cardiomyocyte lineage. Elevated NHE1 expression appears to be triggered as part of the process that facilitates cardiomyocyte development.

Hedgehog is required for murine yolk sac angiogenesis

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 361-372 ◽  
Author(s):  
Noah Byrd ◽  
Sandy Becker ◽  
Peter Maye ◽  
Roopa Narasimhaiah ◽  
Benoit St-Jacques ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Vascular Remodeling ◽  
Hedgehog Signaling ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh–/– mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and α-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh–/– lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or Smo. Whereas Ihh–/– yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo–/– yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh–/– yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.

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.

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro

Development ◽  
1995 ◽  
Vol 121 (11) ◽  
pp. 3877-3888 ◽  
Author(s):  
C. Soudais ◽  
M. Bielinska ◽  
M. Heikinheimo ◽  
C.A. MacArthur ◽  
N. Narita ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Targeted Mutagenesis ◽  
Clonal Variation ◽  
Chimeric Mouse ◽  
Visceral Endoderm

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1105-1114 ◽  
Author(s):  
F. Poirier ◽  
C.T. Chan ◽  
P.M. Timmons ◽  
E.J. Robertson ◽  
M.J. Evans ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Embryonic Stem Cell Differentiation ◽  
Cdna Clones ◽  
Stage Of Development ◽  
Before And After

The differentiation in vitro of murine embryonic stem cells to embryoid bodies mimics events that occur in vivo shortly before and after embryonic implantation. We have used this system, together with differential cDNA cloning, to identify genes the expression of which is regulated during early embryogenesis. Here we describe the isolation of several such cDNA clones, one of which corresponds to the gene H19. This gene is activated in extraembryonic cell types at the time of implantation, suggesting that it may play a role at this stage of development, and is subsequently expressed in all of the cells of the mid-gestation embryo with the striking exception of most of those of the developing central and peripheral nervous systems. After birth, expression of this gene ceases or is dramatically reduced in all tissues.

Deficiency in GATA-4 or GATA-6 Diminishes Definitive Hematopoiesis in Murine Embryonic Stem Cell Derived Embryoid Bodies.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2230-2230
Author(s):  
Monique S. Pierre ◽  
Mervin Yoder
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Vegf Receptor ◽  
Visceral Endoderm ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Erythroid Progenitor ◽  
Blood Island

Abstract Formation of mesoderm derived blood islands in the mouse embryonic yolk sac requires the presence of visceral endoderm (VE) and VE derived factors. Murine embryonic stem (ES) cells can be differentiated into embryoid bodies (EBs) which serve as an in vitro model recapitulating many embryonic developmental processes, including formation of early hematopoietic cells. Previous investigators have reported that differentiation of ES cells deficient in either GATA-4 or GATA-6 results in EBs with disrupted differentiation of visceral endoderm and defective blood island formation. In the current study, we have compared GATA-4 and GATA-6 null ES cell derived EBs to wild-type EBs in their ability to commit to early hematopoietic lineages using hematopoietic progenitor colony assays, and used RT-PCR to assess the expression of endoderm genes. As expected, we observed differences in expression of endoderm genes in wild-type and GATA-4 or GATA-6 null EBs. Blast colony forming cell assays and primitive erythroid progenitor assays revealed no difference in the ability of wild-type and GATA-4 or GATA-6 null EBs to form hemangioblast or primitive erythroid progenitor colonies. In contrast, comparisons of definitive hematopoietic progenitor colonies from day 8, 9 and 10 GATA-4 and GATA-6 null EBs revealed a significant reduction in colony numbers at day 8 (p-values < 0.05) compared to wild-type. Strikingly, definitive progenitor colony numbers are rescued nearly to wild-type levels after the addition of the visceral endoderm derived factor vascular endothelial growth factor (VEGF) during EB differentiation. Furthermore, this rescue response can be blocked by the addition of soluble Flk-1 (VEGF receptor) to EB cultures. These results suggest that GATA-4 and GATA-6 transcription factors and/or visceral endoderm are not necessary for hemangioblast, primitive erythroid, or definitive progenitor emergence from EBs but play a role in definitive progenitor expansion in EBs.

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types

2001 ◽  
Vol 13 (1) ◽  
pp. 15 ◽  
Author(s):  
J. Rathjen ◽  
S. Dunn ◽  
M. D. Bettess ◽  
P. D. Rathjen
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Cell Aggregates ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Pluripotent Cells ◽  
Developmental Potential

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2074-2080 ◽  
Author(s):  
Rebecca J. Chan ◽  
Scott A. Johnson ◽  
Yanjun Li ◽  
Mervin C. Yoder ◽  
Gen-Sheng Feng
Keyword(s):  
Cell Differentiation ◽  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Initial Step ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell

Abstract Homozygous mutant (Shp-2Δ46-110) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2Δ46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory factor (LIF)–stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2Δ46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and hemangioblasts was lower in Shp-2Δ46-110 cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2Δ46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Δ46-110 ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.

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