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.

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats

2002 ◽  
Vol 92 (1) ◽  
pp. 288-296 ◽  
Author(s):  
Jiang-Yong Min ◽  
Yinke Yang ◽  
Kimber L. Converso ◽  
Lixin Liu ◽  
Qin Huang ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Transplantation ◽  
Troponin I ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Control Group ◽  
Positive Staining ◽  
Es Cell ◽  
Intramyocardial Injection

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric α-actin, cardiac α-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.

scholarly journals Nodal secures pluripotency upon embryonic stem cell progression from the ground state

2016 ◽  
Author(s):  
Carla Mulas ◽  
Tüzer Kalkan ◽  
Austin Smith
Keyword(s):  
Ground State ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Lineage Specification ◽  
Cell Identity ◽  
Neural Fate ◽  
Cell Progression ◽  
Adherent Culture ◽  
Kinetics Of

SUMMARYNaïve mouse embryonic stem (ES) cells can readily acquire specific fates, but the cellular and molecular processes that enable lineage specification are poorly characterised. Here we investigated progression from the ES cell ground state in adherent culture. We utilised down-regulation of Rex1::GFPd2 to track loss of ES cell identity. We found that cells that have newly down-regulated this reporter have acquired competence for germline induction. They can also be efficiently specified for different somatic lineages, responding more rapidly than naïve cells to inductive cues. Nodal is a candidate autocrine regulator of pluripotency. Abrogation of Nodal signalling did not substantially alter kinetics of exit from the ES cell state, but accelerated subsequent adoption of neural fate at the expense of other lineages. This effect was evident if Nodal was inhibited prior to extinction of ES cell identity. We suggest that Nodal is pivotal for non-neural competence in cells departing naïve pluripotency.

scholarly journals Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period

2020 ◽  
Author(s):  
Stanley E Strawbridge ◽  
Guy B Blanchard ◽  
Austin Smith ◽  
Hillel Kugler ◽  
Graziano Martello
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Identity ◽  
Daughter Cells ◽  
Population Dynamics Model ◽  
Developmental Progression ◽  
Lag Period

ABSTRACTMouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.

scholarly journals Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells

Blood ◽  
2011 ◽  
Vol 117 (15) ◽  
pp. e142-e150 ◽  
Author(s):  
Motohiko Oshima ◽  
Mitsuhiro Endoh ◽  
Takaho A. Endo ◽  
Tetsuro Toyoda ◽  
Yaeko Nakajima-Takagi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Hematopoietic Stem ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome

Abstract Forced expression of the transcription factor HoxB4 has been shown to enhance the self-renewal capacity of mouse bone marrow hematopoietic stem cells (HSCs) and confer a long-term repopulating capacity to yolk sac and embryonic stem (ES) cell–derived hematopoietic precursors. The fact that ES cell–derived precursors do not repopulate bone marrow without HoxB4 underscores an important role for HoxB4 in the maturation of ES-derived hematopoietic precursors into long-term repopulating HSCs. However, the precise molecular mechanism underlying this process is barely understood. In this study, we performed a genome-wide analysis of HoxB4 using ES cell–derived hematopoietic stem/progenitor cells. The results revealed many of the genes essential for HSC development to be direct targets of HoxB4, such as Runx1, Scl/Tal1, Gata2, and Gfi1. The expression profiling also showed that HoxB4 indirectly affects the expression of several important genes, such as Lmo2, Erg, Meis1, Pbx1, Nov, AhR, and Hemgn. HoxB4 tended to activate the transcription, but the down-regulation of a significant portion of direct targets suggested its function to be context-dependent. These findings indicate that HoxB4 reprograms a set of key regulator genes to facilitate the maturation of developing HSCs into repopulating cells. Our list of HoxB4 targets also provides novel candidate regulators for HSCs.

scholarly journals A genome-wide RNAi screen in mouse embryonic stem cells identifies Mp1 as a key mediator of differentiation

2011 ◽  
Vol 208 (13) ◽  
pp. 2675-2689 ◽  
Author(s):  
Bart A. Westerman ◽  
A. Koen Braat ◽  
Nicole Taub ◽  
Marko Potman ◽  
Joseph H.A. Vissers ◽  
...  
Keyword(s):  
Germ Cell ◽  
Es Cells ◽  
Cell Cancer ◽  
Germ Cell Tumors ◽  
Embryonic Stem ◽  
Scaffolding Protein ◽  
Extrinsic Factors ◽  
Genome Wide ◽  
A Genome ◽  
Differentiation And Proliferation

Despite intense investigation of intrinsic and extrinsic factors that regulate pluripotency, the process of initial fate commitment of embryonic stem (ES) cells is still poorly understood. We used a genome-wide short hairpin RNA screen in mouse ES cells to identify genes that are essential for initiation of differentiation. Knockdown of the scaffolding protein Mek binding protein 1 (Mp1, also known as Lamtor3 or Map2k1ip1) stimulated self-renewal of ES cells, blocked differentiation, and promoted proliferation. Fibroblast growth factor 4 (FGF4) signaling is required for initial fate commitment of ES cells. Knockdown of Mp1 inhibited FGF4-induced differentiation but did not alter FGF4-driven proliferation. This uncoupling of differentiation and proliferation was also observed when oncogenic Ras isoforms were overexpressed in ES cells. Knockdown of Mp1 redirected FGF4 signaling from differentiation toward pluripotency and up-regulated the pluripotency-related genes Esrrb, Rex1, Tcl1, and Sox2. We also found that human germ cell tumors (GCTs) express low amounts of Mp1 in the invasive embryonic carcinoma and seminoma histologies and higher amounts of Mp1 in the noninvasive carcinoma in situ precursor and differentiated components. Knockdown of Mp1 in invasive GCT cells resulted in resistance to differentiation, thereby showing a functional role for Mp1 both in normal differentiation of ES cells and in germ cell cancer.

scholarly journals Changes in the Distributions and Dynamics of Polycomb Repressive Complexes during Embryonic Stem Cell Differentiation

2008 ◽  
Vol 28 (9) ◽  
pp. 2884-2895 ◽  
Author(s):  
Xiaojun Ren ◽  
Claudius Vincenz ◽  
Tom K. Kerppola
Keyword(s):  
Cell Differentiation ◽  
Protein Localization ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Bimolecular Fluorescence Complementation ◽  
Global Changes ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
A Genome

ABSTRACT Polycomb group (PcG) transcription regulatory proteins maintain cell identity by sustained repression of numerous genes. The differentiation of embryonic stem (ES) cells induces a genome-wide shift in PcG target gene expression. We investigated the effects of differentiation and protein interactions on CBX family PcG protein localization and dynamics by using fluorescence imaging. In mouse ES cells, different CBX proteins exhibited distinct distributions and mobilities. Most CBX proteins were enriched in foci known as Polycomb bodies. Focus formation did not affect CBX protein mobilities, and the foci dispersed during ES cell differentiation. The mobilities of CBX proteins increased upon the induction of differentiation and decreased as differentiation progressed. The deletion of the chromobox, which mediates interactions with RING1B, prevented the immobilization of CBX proteins. In contrast, the deletion of the chromodomain, which can bind trimethylated lysine 27 of histone H3, had little effect on CBX protein dynamics. The distributions and mobilities of most CBX proteins corresponded to those of CBX-RING1B complexes detected by using bimolecular fluorescence complementation analysis. Epigenetic reprogramming during ES cell differentiation is therefore associated with global changes in the subnuclear distributions and dynamics of CBX protein complexes.

scholarly journals The transcriptionally permissive chromatin state of ES cells is acutely tuned to translational output

2017 ◽  
Author(s):  
Aydan Bulut-Karslioglu ◽  
Trisha A. Macrae ◽  
Juan A. Oses-Prieto ◽  
Sergio Covarrubias ◽  
Michelle Percharde ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Rna Polymerase Ii ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cellular Growth ◽  
Open Chromatin ◽  
Histone Genes ◽  
Scale Analysis ◽  
Genome Wide ◽  
A Genome

SUMMARYA permissive chromatin environment coupled to hypertranscription is critical to drive the rapid proliferation of Embryonic Stem (ES) cells and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ES cells. The results reveal that activity of cellular growth pathways, prominently protein synthesis, perpetuates the euchromatic state and hypertranscription of ES cells. Acute, mild inhibition of translation results in rapid depletion of euchromatic marks in ES cells and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Remarkably, reduced translational output leads to rewiring of open chromatin within 3 hours, including decreased accessibility at a subset of active developmental enhancers and increased accessibility at histone genes and transposable elements. Using a proteome-scale analysis, we show that several euchromatin regulators are unstable proteins and thus continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription and translation sets the pace of proliferation at peri-implantation and may be employed generally by stem/progenitor cells.

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

ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

2013 ◽  
Vol 25 (2) ◽  
pp. 446 ◽  
Author(s):  
Ruchi Sharma ◽  
Aman George ◽  
Manmohan S. Chauhan ◽  
Suresh Singla ◽  
Radhey S. Manik ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Es Cells ◽  
Single Cells ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Rock Inhibitor

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50–80. Y-27632 increased mean colony area (P < 0.05) although it did not improve their survival. It decreased OCT4 expression (P < 0.05), increased NANOG expression (P < 0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P < 0.05) and decreased that of pro-apoptotic genes BAX and BID (P < 0.05). It increased plating efficiency of single-cell suspensions of ES cells (P < 0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P < 0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1–60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

scholarly journals Production of Mice Entirely Derived from Embryonic Stem (ES) Cell with Many Passages by Coculture of ES Cells with Cytochalasin B Induced Tetraploid Embryos.

1995 ◽  
Vol 44 (3) ◽  
pp. 205-210 ◽  
Author(s):  
Otoya UEDA ◽  
Kouichi JISHAGE ◽  
Nobuo KAMADA ◽  
Satomi UCHIDA ◽  
Hiroshi SUZUKI
Keyword(s):  
Cytochalasin B ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell
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