The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Carrie A. Adelman ◽  
Subrata Chattopadhyay ◽  
James J. Bieker
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Smad Pathway ◽  
Cellular Expression ◽  
Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

Specificity of Smad Signaling during Primitive Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2785-2785
Author(s):  
Brian T. Zafonte ◽  
Tara L. Huber ◽  
Gordon Keller ◽  
Todd Evans
Keyword(s):  
Embryoid Body ◽  
Biological Activities ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Dominant Negative ◽  
Hematopoietic Progenitors ◽  
Transcript Levels ◽  
Wide Range ◽  
Primitive Erythropoiesis

Abstract Bone morphogenetic proteins (BMPs) comprise a sub-family of TGF-beta-like molecules that exert a wide range of biological activities during development, and are essential for normal hematopoiesis. However, the precise stage in development that BMP signaling regulates hematopoiesis is not defined. Three proteins, Smad1, Smad5, and Smad8 transmit BMP signals to the nucleus to activate the expression of hematopoietic-specific transcription factors. These Smads are homologous in their sequences, and appear to be regulated similarly, however their specificity in regulating hematopoiesis remains undefined. Although Smad proteins are regulated post-translationally, their expression is also under transcriptional control during development. We examined the specificity of Smad1/5/8 activity in the context of primitive erythropoiesis, using the mouse embryonic stem cell /embryoid body (ES/EB) system. We exploited ES cells with GFP targeted to the brachyury locus, in order to identify specific sub-sets of progenitors. Smad1 transcript levels are initially upregulated as ES cells become fated to mesoderm and hematopoietic progenitors, but the levels are significantly decreased in cells derived from differentiating primitive erythroid colonies. In contrast, Smad5 transcript levels show the opposite profile, being more correlated with erythroid differentiation. To directly assess the role of these Smads during erythropoiesis, their activity is being manipulated in ES cells during the commitment phases of embryonic hematopoiesis. For this purpose, inducible ES cell lines were generated capable of forcing the expression of wildtype Smad1 or Smad5, or a dominant-negative isoform of Smad5, at any stage of ES/EB development. Colony assays were used to analyze quantitatively the hematopoietic potential of these cells. Forced expression of Smad1 results in a marked increase in primitive red blood cell colony formation as compared to control ES cells. Maintenance of Smad1 expression does not appear to inhibit terminal differentiation. Based on a time-study of the induction, the effect on erythoid colonies could be due to expansion of earlier progenitors. Current experiments using the in vitro blast assay are examining the direct effect of Smad1 expression on earlier (hemangioblast) development. This data, and analogous analyses of cells induced to express Smad5 or the dominant-negative Smad isoform are in progress and will be presented. These studies should facilitate our understanding of the specificity of BMP-regulated Smads during commitment and differentiation of embryonic stem cells and hematopoietic progenitors.

Tyrosine phosphatase SHP-1 acts at different stages of development to regulate hematopoiesis

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4290-4297 ◽  
Author(s):  
Nicholas R. D. Paling ◽  
Melanie J. Welham
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Dominant Negative ◽  
Body Development ◽  
Undifferentiated Cells ◽  
Hematopoietic Precursor ◽  
Multiple Stages

Abstract Mice lacking SHP-1 exhibit a plethora of perturbations in their hematopoietic and immune systems. To reveal the primary effects resulting from SHP-1 deficiency, we used embryonic stem (ES) cells to study the role of SHP-1 in developmental hematopoiesis. We expressed wild-type (WT) and dominant-negative (R459M) forms of SHP-1 in ES cells and used ES/OP-9 coculture and embryoid body development followed by hematopoietic colony assays to demonstrate that SHP-1 acts at multiple stages of hematopoietic differentiation to alter lineage balance. Expression of WT SHP-1 reduced myeloid colony numbers while increasing the numbers of secondary embryoid bodies and mixed hematopoietic colonies obtained. Conversely, expression of R459M SHP-1 resulted in a significant increase in the numbers and sizes of myeloid colonies observed while reducing the numbers of colonies derived from undifferentiated cells or hematopoietic precursor cells. Confining the expression of WT or R459M SHP-1 to the early phases of differentiation decreased and increased progenitor cell numbers, respectively, and influenced colony formation. Overall, our results are consistent with SHP-1 acting during multiple stages of hematopoietic development, and they suggest that the increases in granulocytes and macrophages observed in motheaten mice arise as the result of a cell autonomous effect early during development.

Abstract 1017: Post-translational Modification Of GATA-4 Involved In The Differentiation Of Monkey ES Cell Into Cardiac Myocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
mohsen hosseinkhani ◽  
Hossein Hosseinkhani ◽  
Ali Khademhosseini
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cardiac Myocytes ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Left Ventricular ◽  
Specific Gene ◽  
Es Cell ◽  
Post Translational Modification

Transplantation of embryonic stem (ES) cells into infracted myocardium has been shown to preserve left ventricular function in rodents. Before application of ES cell therapy in humans, however, it is critical to perform pre-clinical studies in large animals such as primates. Characteristics of cynomolgus monkey ES cells are similar with those of human ES cells, but quite different from those of mouse ES cells. Differentiation of Embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetytrans-ferases (HATs) and Histone deactylases (HDACs) govern gene expression patterns by being recruited to the target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for actylation and DNA binding of GATA-4 and its full transcription activity as well as for promotion of a transcriptionally active chromatin configuration. The role of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of monkey ES cells into cardiac myocytes remained unknown. In an ES cell model of developing embryonic bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. TSA facilitate the expression of endogenous cardiac β-myosing heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histone are involved in the differentiation of monkey ES cells into cardiac myocytes.

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.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

scholarly journals Improved Experimental Procedures for Achieving Efficient Germ Line Transmission of Nonobese Diabetic (NOD)-Derived Embryonic Stem Cells

2004 ◽  
Vol 5 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Satoko Arai ◽  
Christina Minjares ◽  
Seiho Nagafuchi ◽  
Toru Miyazaki
Keyword(s):  
High Efficiency ◽  
Germ Line ◽  
Gene Knockout ◽  
Es Cells ◽  
Embryonic Stem ◽  
Nod Mice ◽  
Insulin Dependent Diabetes Mellitus ◽  
Transmission Efficiency ◽  
Dependent Diabetes Mellitus ◽  
Specific Gene

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g.,Iddlocus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBSLett., 455, 101–104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a “pure” NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.

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.

scholarly journals Bivalent genes that undergo transcriptional switching identify networks of key regulators of embryonic stem cell differentiation

BMC Genomics ◽  
2020 ◽  
Vol 21 (S10) ◽  
Author(s):  
Ah-Jung Jeon ◽  
Greg Tucker-Kellogg
Keyword(s):  
Cell Differentiation ◽  
Time Series Data ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Chromatin State ◽  
Series Data ◽  
Embryonic Stem Cell Differentiation ◽  
Gene Promoters

Abstract Background Bivalent promoters marked with both H3K27me3 and H3K4me3 histone modifications are characteristic of poised promoters in embryonic stem (ES) cells. The model of poised promoters postulates that bivalent chromatin in ES cells is resolved to monovalency upon differntiation. With the availability of single-cell RNA sequencing (scRNA-seq) data, subsequent switches in transcriptional state at bivalent promoters can be studied more closely. Results We develop an approach for capturing genes undergoing transcriptional switching by detecting ‘bimodal’ gene expression patterns from scRNA-seq data. We integrate the identification of bimodal genes in ES cell differentiation with analysis of chromatin state, and identify clear cell-state dependent patterns of bimodal, bivalent genes. We show that binarization of bimodal genes can be used to identify differentially expressed genes from fractional ON/OFF proportions. In time series data from differentiating cells, we build a pseudotime approximation and use a hidden Markov model to infer gene activity switching pseudotimes, which we use to infer a regulatory network. We identify pathways of switching during differentiation, novel details of those pathway, and transcription factor coordination with downstream targets. Conclusions Genes with expression levels too low to be informative in conventional scRNA analysis can be used to infer transcriptional switching networks that connect transcriptional activity to chromatin state. Since chromatin bivalency is a hallmark of gene promoters poised for activity, this approach provides an alternative that complements conventional scRNA-seq analysis while focusing on genes near the ON/OFF boundary of activity. This offers a novel and productive means of inferring regulatory networks from scRNA-seq data.

398 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES BY USING SLOW TURNING LATERAL VESSEL (STLV/BIOREACTOR)

2010 ◽  
Vol 22 (1) ◽  
pp. 355
Author(s):  
S. Rungarunlert ◽  
K. Tar ◽  
S. Muenthaisong ◽  
M. Techakumphu ◽  
M. Pirity ◽  
...  
Keyword(s):  
Large Scale ◽  
Embryoid Body ◽  
Troponin T ◽  
Es Cells ◽  
Statistical Significance ◽  
Embryonic Stem ◽  
Industrial Applications ◽  
Cardiac Differentiation ◽  
Seeding Density ◽  
Es Cell

Cardiomyocytes derived from embryonic stem (ES) cells are anticipated to be valuable for cardiovascular drug testing and disease therapies. The overall efficiency and quantity of cardiomyocytes obtained by differentiation of ES cells is still low. To enable a large-scale culture of ES-derived cells, we have tested a scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, bioreactor/STLV (slow turning lateral vessel, Synthecon, Inc., Houston, TX, USA) following inoculation with a single cell suspension of mouse ES cells. Technical parameters for optimal cell expansion and efficient ES cell differentiation were compared, such as ES cell seeding density (3 × 105 and 5 × 105 cells mL-1) into the bioreactor and day of transfer and plating of EB on gelatinated petri dishes (Day 2, Day 3, Day 4, and Day 5). The quantity and quality of EB production including the yield and size of EB, as well as viability and apoptosis of cells, were analyzed. Furthermore, after cultivation, well-developed contracting EB with functional cardiac muscle were obtained in which the percentage of EB beating/well and several specific cardiac genes [cardiac Troponin T (cTnT) and α-actinin] expression were also determined. Data are expressed as mean ± SEM of at least 3 independent experiments. Statistical analyses included one-way ANOVA and Student’s t-test Statistical significance was set at P < 0.05. The results showed that 5 × 105 ES cells mL-1 seeded into the STLV significantly improved the homogeneity of size of EB formed compared with 3 × 105 ES cells mL-1. The EB derived from Days 2 or 3 culturing in STLV had less necrotic cells than Days 4 and 5 groups. Furthermore, plating these EB on Days 2 and 3 resulted in significantly more EB beating/well than that of Days 4 and 5 groups. For cardiac differentiation, the group with 5 × 105 ES cells mL-1 seeded into STLV and transferred and plated on Day 3 expressed more cardiac markers than other groups. In conclusion, the optimized rotary suspension culture method can produce a highly uniform population of efficiently differentiating EB in large quantities in a manner that can be easily implemented by basic research laboratories. This method provides a technological platform for the controlled large-scale generation of ES cell-derived cells for clinical and industrial applications. This work was financed by The Thailand Commission on Higher Education (CHE-PhD-SW-2005-100), EUFP6 CLONET (MRTN-CT-2006-035468), NKFP_07_1-ES2HEART-HU (OM-00202-2007), and EUFP7 (PartnErS, PIAP-GA-2008-218205).

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