scholarly journals Increased Apoptosis and Skewed Differentiation in Mouse Embryonic Stem Cells Lacking the Histone Methyltransferase Mll2

2007 ◽  
Vol 18 (6) ◽  
pp. 2356-2366 ◽  
Author(s):  
Sandra Lubitz ◽  
Stefan Glaser ◽  
Julia Schaft ◽  
A. Francis Stewart ◽  
Konstantinos Anastassiadis
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Lineage Commitment ◽  
Es Cell ◽  
Histone 3 ◽  
Caspase Inhibition ◽  
Transcriptional Memory

Epigenetic regulation by histone methyltransferases provides transcriptional memory and inheritable propagation of gene expression patterns. Potentially, the transition from a pluripotent state to lineage commitment also includes epigenetic instructions. The histone 3 lysine 4 methyltransferase Mll2/Wbp7 is essential for embryonic development. Here, we used embryonic stem (ES) cell lines deficient for Mll2 to examine its function more accurately. Mll2−/− ES cells are viable and retain pluripotency, but they display cell proliferation defects due to an enhanced rate of apoptosis. Apoptosis was not relieved by caspase inhibition and correlated with decreased Bcl2 expression. Concordantly, Mll2 binds to the Bcl2 gene and H3K4me3levels are reduced at the binding site when Mll2 is absent. In vitro differentiation showed delays along representative pathways for all three germ layers. Although ectodermal delays were severe and mesodermal delays persisted at about three days, endodermal differentiation seemed to recover and overshoot, concomitant with prolonged Oct4 gene expression. Hence, Mll2 is not required for ES cell self-renewal or the complex changes in gene expression involved in lineage commitment, but it contributes to the coordination and timing of early differentiation decisions.

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

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

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.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

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.

Development of a Testing Strategy for Detecting Embryotoxic Hazards of Chemicals In Vitro by using Embryonic Stem Cell Models

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 107-109 ◽  
Author(s):  
Susanne Bremer ◽  
Cristian Pellizzer ◽  
Sarah Adler ◽  
Martin Paparella ◽  
Jan de Lange
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Tests ◽  
Es Cell ◽  
Cell Models ◽  
Testing Strategy ◽  
Chemical Effects ◽  
Cell Test ◽  
Stem Cell Models

The importance of developing in vitro tests for embryotoxicity is discussed, and ECVAM's work with its collaborators is summarised. Studies are in progress to find new endpoints for use in the scientifically validated embryonic stem (ES) cell test, so that the potential for chemical effects on endodermal, mesodermal and/or ectodermal differentiation can be identified. This involves, inter alia, the use of genetically modified ES cells.

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.

Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4622-4631 ◽  
Author(s):  
William L. Stanford ◽  
Georgina Caruana ◽  
Katherine A. Vallis ◽  
Maneesha Inamdar ◽  
Michihiro Hidaka ◽  
...  
Keyword(s):  
Large Scale ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Novel Genes ◽  
Specific Expression ◽  
Blood Island

Abstract We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

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.

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.

Sign in / Sign up

Export Citation Format

Share Document