scholarly journals Loss of PRC2 subunits primes lineage choice during exit of pluripotency

2020 ◽  
Author(s):  
Chet H Loh ◽  
Matteo Perino ◽  
Magnus R Bark ◽  
Gert Jan C Veenstra
Keyword(s):  
Es Cells ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
List Type ◽  
Cell Fates ◽  
Germ Layers ◽  
Polycomb Repressive Complex 2 ◽  
Lineage Priming

AbstractPolycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. There are two distinct PRC2 complexes, PRC2.1 and PRC2.2, which contain respectively MTF2 and JARID2 in ES cells. Very little is known about the roles of these auxiliary PRC2 subunits during the exit of pluripotency. In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null embryonic stem cells (ESCs). We observed that the loss of Mtf2 resulted in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells were predominantly directed towards early differentiating precursors and neuro-ectodermal fates. Interestingly, we found that these effects are caused by derepression of developmental regulators that were poised for activation in pluripotent cells and gained H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories were relatively similar to those of wild type cells. Together, our results uncovered a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, providing a threshold for lineage choice during the exit of pluripotency.HighlightsEnhanced and faster differentiation into all three germ layers in Mtf2 null embryoid bodiesJarid2 null cells enriched for early differentiating precursors and neuro-ectodermal cell fatesMTF2 is critical for the balance of activation and repression of key developmental regulatorsPRC2 coordinates lineage choice and execution of the lineage-specific program by thresholding of lineage-priming

scholarly journals Loss of PRC2 subunits primes lineage choice during exit of pluripotency

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chet H. Loh ◽  
Siebe van Genesen ◽  
Matteo Perino ◽  
Magnus R. Bark ◽  
Gert Jan C. Veenstra
Keyword(s):  
Gene Activation ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Specific Gene ◽  
Cell Fates ◽  
Lineage Specification ◽  
Polycomb Repressive Complex 2 ◽  
Expression Of Genes ◽  
Coordinated Expression ◽  
Lineage Specific Gene

AbstractPolycomb Repressive Complex 2 (PRC2) is crucial for the coordinated expression of genes during early embryonic development, catalyzing histone H3 lysine 27 trimethylation. Two distinct PRC2 complexes, PRC2.1 and PRC2.2, contain respectively MTF2 and JARID2 in embryonic stem cells (ESCs). In this study, we explored their roles in lineage specification and commitment, using single-cell transcriptomics and mouse embryoid bodies derived from Mtf2 and Jarid2 null ESCs. We observe that the loss of Mtf2 results in enhanced and faster differentiation towards cell fates from all germ layers, while the Jarid2 null cells are predominantly directed towards early differentiating precursors, with reduced efficiency towards mesendodermal lineages. These effects are caused by derepression of developmental regulators that are poised for activation in pluripotent cells and gain H3K4me3 at their promoters in the absence of PRC2 repression. Upon lineage commitment, the differentiation trajectories are relatively similar to those of wild-type cells. Together, our results uncover a major role for MTF2-containing PRC2.1 in balancing poised lineage-specific gene activation, whereas the contribution of JARID2-containing PRC2 is more selective in nature compared to MTF2. These data explain how PRC2 imposes thresholds for lineage choice during the exit of pluripotency.

scholarly journals Tracking the embryonic stem cell transition from ground state pluripotency

2016 ◽  
Author(s):  
Tüzer Kalkan ◽  
Nelly Olova ◽  
Mila Roode ◽  
Carla Mulas ◽  
Heather J. Lee ◽  
...  
Keyword(s):  
Ground State ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
List Type ◽  
Es Cell ◽  
Genome Wide ◽  
Developmental Progression ◽  
A Genome ◽  
Lineage Priming

SummaryMouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.HighlightsThe Rex1 destabilized GFP reporter demarcates naive pluripotency.Exit from the naive state is asynchronous in the population.Transition is relatively acute in individual cells and precedes lineage priming.Transcriptome and DNA methylome reflect events in the pre-gastrulation embryo.

Human embryonic stem cells: prototypical pluripotential progenitors

2002 ◽  
Vol 10 (3) ◽  
pp. 187-199 ◽  
Author(s):  
R Mollard ◽  
BJ Conley ◽  
AO Trounson
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Germ Layers ◽  
Mouse Es Cells ◽  
Morphological Criteria ◽  
Human Es Cells

Embryonic stem (ES) cells are a primitive cell type derived from the inner cell mass (ICM) of the developing embryo. When cultured for extended periods, ES cells maintain a high telomerase activity, normal karyotype and the pluripotential developmental capacity of their ICM derivatives. Such capacity is best demonstrated by mouse ES cells which can contribute to all tissues of the developing embryo following either their injection into host blastocysts or tetraploid embryo complimentation (for a review see Robertson). For both practical and ethical reasons it is not possible to inject human ES cells into blastocysts for the development of a term fetus. However, when injected beneath the testicular capsule of severe combined immunodeficient (SCID) mice, human ES cells form teratomas comprising tissue representatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) thus attesting to their pluripotency. Based upon morphological criteria, neuronal, cardiac, bone, squamous epithelium, skeletal muscle, gut and respiratory epithelia are readily identifiable within the human ES-cell-derived teratomas. With the demonstrated capability to isolate and maintain pluripotent human ES cells in vitro, their ability to give rise to tissue representatives of all three embryonic germ layers and the technical advances made possible by research on mouse ES cells, a rapid increase in human ES cell research aimed at drug discovery and human cell and gene therapies has occurred. Indeed in the mouse, dissociated embryoid bodies (EBs) have already been demonstrated capable of repopulating the haematopoietic system of recipient animals (for a review see Keller) and mouse ES cells are currently being used in attempts to repair mouse neural degenerative lesions.

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.

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.

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.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

Tie-1-directed expression of Cre recombinase in endothelial cells of embryoid bodies and transgenic mice

2001 ◽  
Vol 114 (4) ◽  
pp. 671-676 ◽  
Author(s):  
E. Gustafsson ◽  
C. Brakebusch ◽  
K. Hietanen ◽  
R. Fassler
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Cre Recombinase ◽  
Embryoid Bodies ◽  
Lymphoid Cells ◽  
Adult Brain ◽  
Specific Gene

Tissue-specific gene inactivation using the Cre-loxP system has become an important tool to unravel functions of genes when the conventional null mutation is lethal. We report here the generation of a transgenic mouse line expressing Cre recombinase in endothelial cells. In order to avoid the production and screening of multiple transgenic lines we used embryonic stem cell and embryoid body technology to identify recombinant embryonic stem cell clones with high, endothelial-specific Cre activity. One embryonic stem cell clone that showed high Cre activity in endothelial cells was used to generate germline chimeras. The in vivo efficiency and specificity of the transgenic Cre was analysed by intercrossing the tie-1-Cre line with the ROSA26R reporter mice. At initial stages of vascular formation (E8-9), LacZ staining was detected in almost all cells of the forming vasculature. Between E10 and birth, LacZ activity was detected in most endothelial cells within the embryo and of extra-embryonic tissues such as yolk sac and chorioallantoic placenta. Ectopic expression of Cre was observed in approximately 12–20% of the adult erythroid, myeloid and lymphoid cells and in subregions of the adult brain. These results show that the tie-1-Cre transgenic strain can efficiently direct deletion of floxed genes in endothelial cells in vivo.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

2016 ◽  
Vol 16 (1) ◽  
pp. 115-128 ◽  
Author(s):  
Thanh Luan Nguyen ◽  
Jae Gyu Yoo ◽  
Neelesh Sharma ◽  
Sung Woo Kim ◽  
Yong Jun Kang ◽  
...  
Keyword(s):  
Developmental Biology ◽  
Regenerative Medicine ◽  
Connexin 43 ◽  
Es Cells ◽  
Periodic Acid ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Significant Finding ◽  
Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

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