Failure to increase glucose consumption through the pentose-phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected to oxidative stress

Mouse embryonic stem (ES) glucose-6-phosphate (G6P) dehydrogenase-deleted cells (G6pdΔ), obtained by transient Cre recombinase expression in a G6pd-loxed cell line, are unable to produce G6P dehydrogenase (G6PD) protein (EC 1.1.1.42). These G6pdΔ cells proliferate in vitro without special requirements but are extremely sensitive to oxidative stress. Under normal growth conditions, ES G6pdΔ cells show a high ratio of NADPH to NADP+ and a normal intracellular level of GSH. In the presence of the thiol scavenger oxidant, azodicarboxylic acid bis[dimethylamide], at concentrations lethal for G6pdΔ but not for wild-type ES cells, NADPH and GSH in G6pdΔ cells dramatically shift to their oxidized forms. In contrast, wild-type ES cells are able to increase rapidly and intensely the activity of the pentose-phosphate pathway in response to the oxidant. This process, mediated by the [NADPH]/[NADP+] ratio, does not occur in G6pdΔ cells. G6PD has been generally considered essential for providing NADPH-reducing power. We now find that other reactions provide the cell with a large fraction of NADPH under non-stress conditions, whereas G6PD is the only NADPH-producing enzyme activated in response to oxidative stress, which can act as a guardian of the cell redox potential. Moreover, bacterial G6PD can substitute for the human enzyme, strongly suggesting that a relatively simple mechanism of enzyme kinetics underlies this phenomenon.

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Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽

10.1242/dev.116.supplement.157 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 157-165 ◽

Cited By ~ 20

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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Beneficial effects of acute inhibition of the oxidative pentose phosphate pathway in the failing heart

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00783.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. H709-H717 ◽

Cited By ~ 13

Author(s):

Claudio Vimercati ◽

Khaled Qanud ◽

Gianfranco Mitacchione ◽

Danuta Sosnowska ◽

Zoltan Ungvari ◽

...

Keyword(s):

Oxidative Stress ◽

Pentose Phosphate Pathway ◽

Tissue Concentration ◽

Glucose Consumption ◽

Pentose Phosphate ◽

Oxidative Pentose Phosphate Pathway ◽

Stroke Work ◽

Failing Heart

In vitro studies suggested that glucose metabolism through the oxidative pentose phosphate pathway (oxPPP) can paradoxically feed superoxide-generating enzymes in failing hearts. We therefore tested the hypothesis that acute inhibition of the oxPPP reduces oxidative stress and enhances function and metabolism of the failing heart, in vivo. In 10 chronically instrumented dogs, congestive heart failure (HF) was induced by high-frequency cardiac pacing. Myocardial glucose consumption was enhanced by raising arterial glycemia to levels mimicking postprandial peaks, before and after intravenous administration of the oxPPP inhibitor 6-aminonicotinamide (80 mg/kg). Myocardial energy substrate metabolism was measured with radiolabeled glucose and oleic acid, and cardiac 8-isoprostane output was used as an index of oxidative stress. A group of five chronically instrumented, normal dogs served as control. In HF, raising glycemic levels from ∼80 to ∼170 mg/dL increased cardiac isoprostane output by approximately twofold, whereas oxPPP inhibition normalized oxidative stress and enhanced cardiac oxygen consumption, glucose oxidation, and stroke work. In normal hearts glucose infusion did not induce significant changes in cardiac oxidative stress. Myocardial tissue concentration of 6P-gluconate, an intermediate metabolite of the oxPPP, was significantly reduced by ∼50% in treated versus nontreated failing hearts, supporting the inhibitory effect of 6-aminonicotinamide. Our study indicates an important contribution of the oxPPP activity to cardiac oxidative stress in HF, which is particularly pronounced during common physiological changes such as postprandial glycemic peaks.

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Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

Journal of Experimental Medicine ◽

10.1084/jem.191.5.859 ◽

2000 ◽

Vol 191 (5) ◽

pp. 859-870 ◽

Cited By ~ 207

Author(s):

Melanie Allen ◽

Linne Svensson ◽

Marsha Roach ◽

John Hambor ◽

John McNeish ◽

...

Keyword(s):

Map Kinase ◽

Es Cells ◽

Interleukin 1 ◽

Embryonic Stem ◽

Wild Type ◽

Antiinflammatory Drugs ◽

Gene Encoding ◽

Significant Difference ◽

Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

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Thyroid Hormone-Dependent Gene Expression in Differentiated Embryonic Stem Cells and Embryonal Carcinoma Cells: Identification of Novel Thyroid Hormone Target Genes by Deoxyribonucleic Acid Microarray Analysis

Endocrinology ◽

10.1210/en.2004-1177 ◽

2005 ◽

Vol 146 (2) ◽

pp. 776-783 ◽

Cited By ~ 13

Author(s):

Yan-Yun Liu ◽

Gregory A. Brent

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Thyroid Hormone ◽

Early Development ◽

Target Genes ◽

Es Cells ◽

Embryonic Stem ◽

Wild Type ◽

Ec Cells

Abstract T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRα P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.

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Cardiomyocyte differentiation by GATA-4-deficient embryonic stem cells

Development ◽

10.1242/dev.124.19.3755 ◽

1997 ◽

Vol 124 (19) ◽

pp. 3755-3764 ◽

Cited By ~ 4

Author(s):

N. Narita ◽

M. Bielinska ◽

D.B. Wilson

Keyword(s):

Transcription Factor ◽

In Situ Hybridization ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Cardiomyocyte Differentiation ◽

Wild Type ◽

Cell Stage

In situ hybridization studies, promoter analyses and antisense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In this study, we utilized Gata4−/− embryonic stem (ES) cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. First, we assessed the ability of Gata4−/− ES cells form cardiomyocytes during in vitro differentiation of embryoid bodies. Contracting cardiomyocytes were seen in both wild-type and Gata4−/− embryoid bodies, although cardiomyocytes were observed more often in wild type than in mutant embryoid bodies. Electron microscopy of cardiomyocytes in the Gata4−/− embryoid bodies revealed the presence of sarcomeres and junctional complexes, while immunofluorescence confirmed the presence of cardiac myosin. To assess the capacity of Gata4−/− ES cells to differentiate into cardiomyocytes in vivo, we prepared and analyzed chimeric mice. Gata4−/− ES cells were injected into 8-cell-stage embryos derived from ROSA26 mice, a transgenic line that expresses beta-galactosidase in all cell types. Chimeric embryos were stained with X-gal to discriminate ES cell- and host-derived tissue. Gata4−/− ES cells contributed to endocardium, myocardium and epicardium. In situ hybridization showed that myocardium derived from Gata4−/− ES cells expressed several cardiac-specific transcripts, including cardiac alpha-myosin heavy chain, troponin C, myosin light chain-2v, Nkx-2.5/Csx, dHAND, eHAND and GATA-6. Taken together these results indicate that GATA-4 is not essential for terminal differentiation of cardiomyocytes and suggest that additional GATA-binding proteins known to be in cardiac tissue, such as GATA-5 or GATA-6, may compensate for a lack of GATA-4.

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Blunted Reducing Power Generation in Erythrocytes Contributes to Oxidative Stress in Prepubertal Obese Children with Insulin Resistance

Antioxidants ◽

10.3390/antiox10020244 ◽

2021 ◽

Vol 10 (2) ◽

pp. 244

Author(s):

Álvaro González-Domínguez ◽

Francisco Visiedo ◽

Jesus Domínguez-Riscart ◽

Beatriz Ruiz-Mateos ◽

Ana Saez-Benito ◽

...

Keyword(s):

Oxidative Stress ◽

Insulin Resistance ◽

Power Generation ◽

Antioxidant Enzymes ◽

Pentose Phosphate Pathway ◽

Reducing Power ◽

Redox Status ◽

Pentose Phosphate ◽

Obese Children ◽

Insulin Resistant

Childhood obesity, and specifically its metabolic complications, are related to deficient antioxidant capacity and oxidative stress. Erythrocytes are constantly exposed to multiple sources of oxidative stress; hence, they are equipped with powerful antioxidant mechanisms requiring permanent reducing power generation and turnover. Glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) are two key enzymes on the pentose phosphate pathway. Both enzymes supply reducing power by generating NADPH, which is essential for maintaining the redox balance within the cell and the activity of other antioxidant enzymes. We hypothesized that obese children with insulin resistance would exhibit blunted G6PDH and 6PGDH activities, contributing to their erythrocytes’ redox status imbalances. We studied 15 control and 24 obese prepubertal children, 12 of whom were insulin-resistant according to an oral glucose tolerance test (OGTT). We analyzed erythroid malondialdehyde (MDA) and carbonyl group levels as oxidative stress markers. NADP+/NADPH and GSH/GSSG were measured to determine redox status, and NADPH production by both G6PDH and 6PGDH was assayed spectrophotometrically to characterize pentose phosphate pathway activity. Finally, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GR) activities were also assessed. As expected, MDA and carbonyl groups levels were higher at baseline and along the OGTT in insulin-resistant children. Both redox indicators showed an imbalance in favor of the oxidized forms along the OGTT in the insulin-resistant obese group. Additionally, the NADPH synthesis, as well as GR activity, were decreased. H2O2 removing enzyme activities were depleted at baseline in both obese groups, although after sugar intake only metabolically healthy obese participants were able to maintain their catalase activity. No change was detected in SOD activity between groups. Our results show that obese children with insulin resistance present higher levels of oxidative damage, blunted capacity to generate reducing power, and hampered function of key NADPH-dependent antioxidant enzymes.

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Biological Analysis of AML1/RUNX1 Arginine-Mutants by Means of Hematopoietic Rescue Experiments of Runx1-deficient Mouse Embryonic Stem Cells,

Blood ◽

10.1182/blood.v118.21.3382.3382 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3382-3382

Author(s):

Shinsuke Mizutani ◽

Masafumi Taniwaki ◽

Tsukasa Okuda

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Biological Significance ◽

Deficient Mouse ◽

Es Cell ◽

Wild Type ◽

Post Translational Modification ◽

Cell Clones ◽

Arginine Residues

Abstract Abstract 3382 Runt-Related Transcription Factor 1 (RUNX1; also called as Acute Myeloid Leukemia 1: AML1) is one of the most frequently mutated genes associated with human acute leukemia, and encodes DNA binding subunit of the Core-Binding Factor (CBF) transcription complex whose activity is essential for the development of definitive hematopoiesis. RUNX1 serves as a transcriptional activator as well as a repressor to its target genes, depending on the cellular context, mediated through its interaction with co-factors. Increasing evidence obtained these days suggests that post-translational modification of RUNX1, including phosphorylation, methylation, or acetylation on its target amino acid residues, is important for proper and fine tuning of this RUNX1-function, likely by altering its association with functional cofactors. However, biological significance of these modifications has not yet been examined in detail. As an initial effort towards systematic comprehension how these modifications influence RUNX1 function, we tried to evaluate RUNX1 methylation in vitro in this study. Arginine residues just douwnstream to the Runt-domain of RUNX1 were recently reported to be methylated to inhibit corepressor-binding thus enhances its trans-activating activity. In order to elucidate the biological effects of this post-translational modification, we manufactured arginine-to-lysine substitutions at the sites within the mouse cDNA. When these arginine-mutants were exogenously expressed in mammalian cell lines, they showed reduced trans-activating activity detected by a dual-luciferase assay on known reporter constructs in comparison to the wild-type Runx1, confirming previous reports. We then introduced the mutant cDNA into Runx1-deficient mouse embryonic stem (ES) cells by means of a knock-in strategy at the disrupted Runx1 gene locus. These ES cell clones were subjected to the in vitro differentiation to hematopoietic lineages. Wild-type ES cells are known to differentiate into hematopoietic cell lineages via embryoid body formation in a semi-solid culture system, whereas ES cells of Runx1-deficient genotype lose the ability to undergo hematopoietic differentiation. This phenomenon is recognized to be an in vitro phenocopy of the Runx1-deficient mice that suffer from embryonic death due to complete block of fetal liver hematopoiesis. Initial study so far showed that the Runx1-deficient ES cell clones restored the ability to develop hematopoietic cells including macrophages in culture when the arginine-mutant cDNA was re-expressed from the knock-in allele, as is the case for the control Runx1-deficient ES cells with the knocked-in wild-type Runx1. These results suggest that this arginine-to-lysine mutation is dispensable, at least, for the in vitro hematopoietic function of wild-type Runx1 although its trans-activating activity is somewhat impaired. We are currently focusing on introducing this mutation into mouse germ line, and the resultant genome-modified mice should show us the biological significance of the methylation-modification to this important molecule in the context of an entire animal. Disclosures: No relevant conflicts of interest to declare.

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Oct-4 Is Crucial for Murine Embryonic Stem Cell Secretion of Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis of Murine ES Cells and Bone Marrow Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v106.11.1359.1359 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1359-1359

Author(s):

Ying Guo ◽

Barbara Graham-Evans ◽

Charlie R. Mantal ◽

Robert A. Hromas ◽

Hal E. Broxmeyer

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Caspase 3 ◽

Es Cells ◽

Embryonic Stem ◽

Conditional Knockout ◽

Es Cell ◽

Wild Type ◽

Murine Bone Marrow

Abstract Murine embryonic stem (ES) cells may be of potential use for cell replacement and gene therapy. Maintenance of ES cells in an undifferentiated and proliferative state depends on cytokines either secreted by ES cells and/or added to the medium. By understanding the production and release of cytokines in ES cell culture, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that SDF-1/CXCL12, secreted by ES cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells (Guo et al, Stem cells, in press, 2005). To evaluate whether other cytokines were produced by murine ES cells, we generated conditioned medium (CM) from these cells in the presence of LIF, while the ES cells were in an undifferentiated Oct-4 expressing state, and assayed the CM for cytokines, chemokines, and other growth modulatory factors. ES cell CM enhanced survival in vitro of ES cells subjected to delayed addition of serum to ES cell cultures. Without serum, ES cells didn’t grow in low cell density. However, with CM, ES cells formed colonies at about 63% of the growth of the ES cells in the presence of serum. ES cell CM also enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased the rate of apoptosis in murine bone marrow c-kit+lin− cells as assessed by Annexin V assay. Our data showed ES cell CM contained IL-1α, IL-10, IL-11, M-CSF, OSM, SCF, VEGF, as well as a number of chemokines and other proteins. For a number of these proteins, we have already verified that the mRNA for them is expressed in the ES cells. This indicates that ES cells produce and secrete these cytokines. Some of these cytokines are known to have an enhanced survival/antiapoptosis effect on progenitors. IL-6, FGF-9, and TNF-a, which were not detected prior to irradiation of the ES cells, were seen after ES cells were irradiated. Irradiation of the ES cells enhanced release of some proteins and decreased release of others. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for cytokines released from ES cells which supported CFU-GM survival and antiapoptosis. Oct-4 conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. CM from the wild type ES cell line enhanced survival of CFU-GM similar to that of other ES cell lines, while the Oct-4 knockout ES cell line didn’t. These results indicate that release of proteins involved in survival enhancement may be related to Oct-4 expression. We also found that the wild type cell line which expressed Oct-4 didn’t initiate caspase 3 dependent apoptosis after mitotic stress. However ZHBTc4, the Oct-4 deleted cell line demonstrated caspase 3 dependent apoptosis. These results may be of physiological significance, although this has not yet been proven, and suggest the possibility of potential future applicability for use of irradiated ES cells as accessory cells for growth modulation in vitro and in vivo.

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

Blood ◽

10.1182/blood.v110.11.2230.2230 ◽

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.

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