scholarly journals The paralogous hematopoietic regulators Lyl1 and Scl are coregulated by Ets and GATA factors, but Lyl1 cannot rescue the early Scl–/– phenotype

Blood ◽  
2006 ◽  
Vol 109 (5) ◽  
pp. 1908-1916 ◽  
Author(s):  
Wan Y. I. Chan ◽  
George A. Follows ◽  
Georges Lacaud ◽  
John E. Pimanda ◽  
Josette-Renee Landry ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Es Cell ◽  
Hematopoietic Stem ◽  
Mild Phenotype ◽  
Gata Factors ◽  
Ets Factors

Abstract Transcription factors are key regulators of hematopoietic stem cells (HSCs), yet the molecular mechanisms that control their expression are largely unknown. Previously, we demonstrated that expression of Scl/Tal1, a transcription factor required for the specification of HSCs, is controlled by Ets and GATA factors. Here we characterize the molecular mechanisms controlling expression of Lyl1, a paralog of Scl also required for HSC function. Two closely spaced promoters directed expression to hematopoietic progenitor, megakaryocytic, and endothelial cells in transgenic mice. Conserved binding sites required for promoter activity were bound in vivo by GATA-2 and the Ets factors Fli1, Elf1, Erg, and PU.1. However, despite coregulation of Scl and Lyl1 by the same Ets and GATA factors, Scl expression was initiated prior to Lyl1 in embryonic stem (ES) cell differentiation assays. Moreover, ectopic expression of Scl but not Lyl1 rescued hematopoietic differentiation in Scl−/− ES cells, thus providing a molecular explanation for the vastly different phenotypes of Scl−/− and Lyl1−/− mouse embryos. Furthermore, coregulation of Scl and Lyl1 later during development may explain the mild phenotype of Scl−/− adult HSCs.

Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells by Their Genetic Manipulation Using the Third Generation Lentiviral Vector.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 495-495
Author(s):  
Ryo Kurita ◽  
Erika Sasaki ◽  
Takashi Hiroyama ◽  
Tomoko Yokoo ◽  
Yukoh Nakazaki ◽  
...  
Keyword(s):  
Cell Therapy ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Preclinical Studies ◽  
Es Cell ◽  
Hematopoietic Stem

Abstract Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of differentiated ES cells to specific organ has been expected to complete its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy. In this study, we investigated the in vitro and in vivo differentiation of CM ES cells to hematopoietic cells by exogenous gene transfer methods in order to study the feasibility of future gene modified ES cell therapy. First, we tried various in vitro culture conditions including systems using embryoid bodies or co-culturing with stromal cells to induce hematopoietic cells, but the frequency of inducing hematopoietic cells was very low. The expression of CD45 and gata1 could not be detected in both conditions, suggesting that our culture conditions were incomplete for induction of hematopoietic cells from CM ES cells. Next we examined gene transduction methods by using VSV-G pseudotyped human immunodeficiency virus (HIV) vectors. We constructed the HIV vectors containing hematopoietic genes such as tal1/scl, gata1, gata2, hoxB4 and Lh2 genes under the EF1a promoter and transduced them into CM ES cells. Only in the case of tal1/scl overexpression, not other genes, hematopoietic induction from CM ES cells was dramatically increased and multi-lineage blood cells consisting of erythroid cells, granulocytes, macrophages and megakaryocytes, were confirmed by immunochemical and morphological analyses. Furthermore, RT-PCR results showed that several hematopoietic marker genes including CD34 were expressed higher in the tal1/scl overexpressed ES-derived cells. After the xenotransplantation of ES-derived cells into the immunodeficient mice, CM CD45+ cells and immature erythroids and megakaryocytic cells were observed only in the ES-tal1-injected mice, indicating that enforced expression of tal1/scl into ES cells led to highly efficient hematopoietic cell differentiation in vivo. Taken together, it was suggested that the transduction of exogenous tal1/scl cDNA into ES cells by HIV vector was the promising method for the efficient differentiation from CM ES cells to hematopoietic stem cells. Further examinations are required to determine the long-term hematopoietic reconstitute capacity and the safety of the tal1/scl transduced ES cells in marmoset for the purpose of developing new hematopoietic stem cell therapy.

scholarly journals Thrombopoietin (TPO) Induces Hematopoietic Differentiation of Mouse ES Cells Via HIF-1 Alpha-Dependent Activation of a BMP4 Autoregulatory Loop

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1186-1186
Author(s):  
Azadeh Zahabi ◽  
Tatsuya Morishima ◽  
Andri Pramono ◽  
Dan Lan ◽  
Lothar Kanz ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Efficient Production ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the optimal conditions for the production of hematopoietic cells as a source for transplantation or experimental use. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c-mpl. This knowledge, together with observations in vitro and in animal models in vivo, suggests that TPO/c-mpl signaling promotes early hematopoiesis. However, the downstream mechanisms underlying TPO signaling are not fully elucidated. Here, we describe for the first time a direct connection between the TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in the hematopoietic fate decision of ES cells. BMP4 is a classical morphogen and a well-known inducer of early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 by ES cells with concomitant upregulation of the BMP receptor, BMPR1A, phosphorylation of Smad1, 5, and 8 and activation of the specific target genes, Id1, 2, and 3, and Msx1 and 2. This was mediated by TPO-dependent binding of the HIF-1α transcription factor to the BMP4 gene promoter, resulting in further activation of the BMP4-autoregulatory loop. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO-dependent hematopoietic differentiation of ES cell. Taken together, our findings contribute to the understanding the mechanisms of hematopoietic differentiaiton of ES cells and might help to establish new methods for the efficient production of hematopoietic stem cells in vitro. Disclosures No relevant conflicts of interest to declare.

Induction and Patterning of Pre-Hemato-Vascular Mesoderm by the Mouse Mix Homeodomain Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 565-565
Author(s):  
Margaret H. Baron ◽  
Stephen Willey ◽  
Kenneth Sahr ◽  
Hailan Zhang ◽  
Kevin Balbi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Target Genes ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryoid Bodies ◽  
Homeodomain Protein ◽  
Stem Cell Markers ◽  
Es Cell ◽  
Hematopoietic Stem

Abstract Members of the Xenopus and zebrafish Mix/Bix family of paired class homeodomain proteins play determining roles in both mesoderm and endoderm development and are induced by members of the TGFbeta/BMP family of signaling molecules. A single Mix gene has been identified in mouse, humans and chick. Prior to gastrulation, the mouse Mix (mMix) gene is expressed in the visceral endoderm and later in the primitive streak and nascent mesoderm, where it overlaps, in part, with T. Mix expression in ES-derived embryoid bodies is early and transient, overlapping partially with Flk1 activation around the time of formation of hemangioblasts. Both mMix mRNA and protein are found in a FACS-sorted population of T+Flk1+ cells from ES cell-derived embryoid bodies (EBs) which contains hemangioblasts. A complex embryonic lethal phenotype has been reported for Mix deficient embryos, including defects in allantoic (vascular) and cardiogenesis. Mesoderm forms in these embryos but is not patterned properly. Embryonic lethality occurs around E10.5–11, presumably as a result of the cardiovascular defects. We have generated inducible ES cell lines in which expression of Mix protein is responsive to doxycycline. Ectopic expression of Mix in EBs results in premature, enhanced expression of hemangioblast, angioblast and hematopoietic stem cell markers (mRNA and FACS analyses) and increased formation of stem/progenitor cells in clonogenic assays in methylcellulose. Together, the expression analyses, knockout phenotype, and gain-of-function studies in ES cells suggest that mMix functions early in induction and patterning of mesoderm, including formation of hematopoietic and endothelial lineages. Potential mMix target genes are being identified by microarray analyses of the inducible Mix ES lines. To examine mMix activities in vivo, we have generated null and conditional mMix knockout mice from several independently targeted ES cell lines. Analysis of these animals is in progress. Like Xenopus Mix.1, mouse Mix may represent an important connection between the TGFbeta/BMP pathway and hematopoietic/vascular development in the embryo.

scholarly journals ImpairedIn VitroErythropoiesis following Deletion of theScl(Tal1) +40 Enhancer Is Largely Compensated forIn Vivodespite a Significant Reduction in Expression

2013 ◽  
Vol 33 (6) ◽  
pp. 1254-1266 ◽  
Author(s):  
Rita Ferreira ◽  
Dominik Spensberger ◽  
Yvonne Silber ◽  
Andrew Dimond ◽  
Juan Li ◽  
...  
Keyword(s):  
Germ Line ◽  
Es Cells ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Erythroid Differentiation ◽  
Erythroid Cell ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Definitive Erythropoiesis

TheScl(Tal1) gene encodes a helix-loop-helix transcription factor essential for hematopoietic stem cell and erythroid development. TheScl+40 enhancer is situated downstream ofMap17, the 3′ flanking gene ofScl, and is active in transgenic mice during primitive and definitive erythropoiesis. To analyze thein vivofunction of theScl+40 enhancer within theScl/Map17transcriptional domain, we deleted this element in the germ line.SclΔ40/Δ40mice were viable with reduced numbers of erythroid CFU in both bone marrow and spleen yet displayed a normal response to stress hematopoiesis. Analysis ofSclΔ40/Δ40embryonic stem (ES) cells revealed impaired erythroid differentiation, which was accompanied by a failure to upregulateSclwhen erythropoiesis was initiated.Map17expression was also reduced in hematopoietic tissues and differentiating ES cells, and theScl+40 element was able to enhance activity of theMap17promoter. However, onlySclbut notMap17could rescue theSclΔ40/Δ40ES phenotype. Together, these data demonstrate that theScl+40 enhancer is an erythroid cell-specific enhancer that regulates the expression of bothSclandMap17. Moreover, deletion of the +40 enhancer causes a novel erythroid phenotype, which can be rescued by ectopic expression ofSclbut notMap17.

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

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

scholarly journals Loss of Tie2 receptor compromises embryonic stem cell–derived endothelial but not hematopoietic cell survival

Blood ◽  
2006 ◽  
Vol 107 (3) ◽  
pp. 1207-1213 ◽  
Author(s):  
Isao Hamaguchi ◽  
Tohru Morisada ◽  
Masaki Azuma ◽  
Kyoko Murakami ◽  
Madoka Kuramitsu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Hematopoietic Cell ◽  
Caspase Inhibitor ◽  
Lymphatic Endothelial Cells ◽  
Es Cell ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Tie2 Receptor

AbstractTie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell–derived Tie2+Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.

258. Differential expression of H2A and H3 variant histones in mouse preimplantation embryos and R1 ES cells

2008 ◽  
Vol 20 (9) ◽  
pp. 58
Author(s):  
G. R. Kafer ◽  
SA Lehnert ◽  
P. L. Kaye ◽  
R. J. Moser
Keyword(s):  
Messenger Rna ◽  
Expression Profiles ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Histone Variants ◽  
Histone Variant ◽  
Preimplantation Embryos ◽  
Es Cell

Histone variants replace canonical histones in nucleosomes to serve numerous biological processes. This integration alters DNA properties to ultimately regulate gene expression. Previous mouse studies have indicated that some variants (H2AZ and H3.3) are essential for survival, but here we document and correlate histone expression patterns with key developmental events. Using quantitative reverse-transcribed PCR (qRT–PCR) we investigated the expression of 7 genes coding for H2A variants and 4 genes coding for H3 variants in mouse preimplantation embryos and in pluripotent R1 ES cells. Messenger RNA was extracted from pools of 3 embryos flushed from superovulated mice. Embryos were collected at five stages, zygotes, 2-cell embryos, morulae, blastocysts and hatching blastocysts (20 h, 44 h, 68 h, 92 h and 116 h post hCG respectively). The expression of H2A variant genes typically peaked within blastocysts. H2AZ and H2AX expression was 80 – 95% higher in blastocysts than other stages. Conversely, genes coding for H3 variants showed elevated expression in zygotes, where H3.3 expression was 85 – 95% higher and CENPA was ~75% higher than in later preimplantation stages. The expression profiles of histone remodellers SWI/SNF and CAF-1 correlated with the variants they are known to remodel (H2A and H3 variants respectively), suggesting an increased integration of those variants into nucleosomes. We also compared blastocyst and embryonic stem cell (ES cell) expression patterns. R1 ES cells express all histone variants, including H2A.Bbd, H3.1 and H3.2 which were not expressed in preimplantation embryos. Further, expression levels of every histone variant investigated differed significantly between R1 ES cells and hatching blastocysts (ANOVA, P < 0.05, n = 3 experiments). We conclude that histone variant expression reflects preimplantation developmental demands. Further, histone code expression profiles show significant change upon extended cell culture and maintenance of pluripotency as indicated by comparing in vivo hatching blastocysts to the R1 ES cell line.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

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