scholarly journals 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 ◽  
2005 ◽  
Vol 146 (2) ◽  
pp. 776-783 ◽  
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.

scholarly journals Tumorigenic and Differentiation Potentials of Embryonic Stem Cells Depend on TGFβFamily Signaling: Lessons from Teratocarcinoma Cells Stimulated to Differentiate with Retinoic Acid

2017 ◽  
Vol 2017 ◽  
pp. 1-14 ◽  
Author(s):  
Olga Gordeeva ◽  
Sergey Khaydukov
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Retinoic Acid ◽  
Es Cells ◽  
Embryonic Stem ◽  
Activin A ◽  
Bmp Signaling ◽  
Induced Differentiation ◽  
Ec Cells

A significant challenge for the development of safe pluripotent stem cell-based therapies is the incomplete in vitro differentiation of the pluripotent stem cells and the presence of residual undifferentiated cells initiating teratoma development after transplantation in recipients. To understand the mechanisms of incomplete differentiation, a comparative study of retinoic acid-induced differentiation of mouse embryonic stem (ES) and teratocarcinoma (EC) cells was conducted. The present study identified differences in proliferative activity, differentiation, and tumorigenic potentials between ES and EC cells. Higher expression of Nanog and Mvh, as well as Activin A and BMP4, was found in undifferentiated ES cells than in EC cells. However, the expression levels of Activin A and BMP4 increased more sharply in the EC cells during retinoic acid-induced differentiation. Stimulation of the Activin/Nodal and BMP signaling cascades and inhibition of the MEK/ERK and PI3K/Act signaling pathways resulted in a significant decrease in the number of Oct4-expressing ES cells and a loss of tumorigenicity, similar to retinoic acid-stimulated EC cells. Thus, this study demonstrates that a differentiation strategy that modulates prodifferentiation and antiproliferative signaling in ES cells may be effective for eliminating tumorigenic cells and may represent a valuable tool for the development of safe stem cell therapeutics.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

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 Differences in Gene Expression between Wild Type and Hoxa1 Knockout Embryonic Stem Cells after Retinoic Acid Treatment or Leukemia Inhibitory Factor (LIF) Removal

2005 ◽  
Vol 280 (16) ◽  
pp. 16484-16498 ◽  
Author(s):  
Eduardo Martinez-Ceballos ◽  
Pierre Chambon ◽  
Lorraine J. Gudas
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Retinoic Acid ◽  
Leukemia Inhibitory Factor ◽  
Target Genes ◽  
Hox Genes ◽  
Es Cells ◽  
Embryonic Stem ◽  
Inhibitory Factor ◽  
Hoxa Cluster

Homeobox (Hox) genes encode a family of transcription factors that regulate embryonic patterning and organogenesis. In embryos, alterations of the normal pattern of Hox gene expression result in homeotic transformations and malformations. Disruption of theHoxa1gene, the most 3′ member of the Hoxa cluster and a retinoic acid (RA) direct target gene, results in abnormal ossification of the skull, hindbrain, and inner ear deficiencies, and neonatal death. We have generated Hoxa1-/-embryonic stem (ES) cells (named Hoxa1-15) from Hoxa1-/-mutant blastocysts to study the Hoxa1 signaling pathway. We have characterized in detail these Hoxa1-/-ES cells by performing microarray analyses, and by this technique we have identified a number of putative Hoxa-1 target genes, including genes involved in bone development (e.g. Col1a1,Postn/Osf2, and the bone sialoprotein gene orBSP), genes that are expressed in the developing brain (e.g. Nnat,Wnt3a,BDNF,RhoB, andGbx2), and genes involved in various cellular processes (e.g. M-RAS,Sox17,Cdkn2b,LamA1,Col4a1,Foxa2,Foxq1,Klf5, andIgf2). Cell proliferation assays and Northern blot analyses of a number of ES cell markers (e.g. Rex1,Oct3/4,Fgf4, andBmp4) suggest that the Hoxa1 protein plays a role in the inhibition of cell proliferation by RA in ES cells. Additionally, Hoxa1-/-ES cells express high levels of various endodermal markers, includingGata4andDab2, and express much lessFgf5after leukemia inhibitory factor (LIF) withdrawal. Finally, we propose a model in which the Hoxa1 protein mediates repression of endodermal differentiation while promoting expression of ectodermal and mesodermal characteristics.

scholarly journals Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Andrea C. Romero ◽  
Eugenio Vilanova ◽  
Miguel A. Sogorb
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
Stem Cell ◽  
Cell Viability ◽  
Embryonic Stem Cell ◽  
Total Duration ◽  
Embryonic Stem ◽  
End Points ◽  
Cell Test

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicityin vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes:Pnpla6,Afp,Hdac7,Vegfa, andNes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression ofNes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting forin vitrorisk assessment embryotoxicity.

scholarly journals c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

2010 ◽  
Vol 30 (6) ◽  
pp. 1329-1340 ◽  
Author(s):  
Ping Xu ◽  
Roger J. Davis
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Self Renewal ◽  
Chemical Genetic ◽  
Specific Differentiation ◽  
Gene Ablation ◽  
Early Embryonic Lethality

ABSTRACT The c-Jun NH2-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1 −/− Jnk2 −/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.

scholarly journals Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

2000 ◽  
Vol 191 (5) ◽  
pp. 859-870 ◽  
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.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 776-776
Author(s):  
Zhongfa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Target Genes ◽  
Embryonic Stem ◽  
Myeloid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

scholarly journals Optimization of the TeraTox assay for preclinical teratogenicity assessment

2021 ◽  
Author(s):  
Manuela Jaklin ◽  
Jitao David Zhang ◽  
Nicole Schaefer ◽  
Nicole Clemann ◽  
Paul Barrow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Machine Learning ◽  
Stem Cell ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Cell Renewal ◽  
Germ Layer

Teratogenicity poses severe threats to patient safety. Stem-cell-based in vitro systems are promising tools to predict human teratogenicity. However, current in vitro assays are limited because they either capture effects on a certain germ layer, or focus on a subset of predictive markers. Here we report the characterization and critical assessment of TeraTox, a newly developed multi-lineage differentiation assay using 3D human induced pluripotent stem cells. TeraTox probes stem-cell derived embryoid bodies with two endpoints, one quantifying cytotoxicity and the other inferring the teratogenicity potential with gene expression as a molecular phenotypic readout. To derive teratogenicity potentials from gene expression profiles, we applied both unsupervised machine-learning tools including factor analysis and supervised tools including classification and regression. To identify the best predictive model for the teratogenicity potential that is explainable, we systematically tested 64 machine-learning model architectures and identified the optimal model, which uses expression of 77 representative germ-layer genes, summarized by 10 latent germ-layer factors, as input for random-forest regression. We combined measured cytotoxicity and inferred teratogenicity potential to predict concentration-dependent teratogenicity profiles of 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Compared with the mouse embryonic stem cell test, which has been in routine use for more than a decade, the TeraTox assay shows higher sensitivity, particularly towards teratogens impairing ectodermal development or stem-cell renewal, and a more balanced prediction performance. We envision that further refinement and development of TeraTox has the potential to reduce and replace animal research in drug discovery and to improve preclinical assessment of teratogenicity.

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