scholarly journals Nanog fluctuations in ES cells highlight the problem of measurement in cell biology

2016 ◽  
Author(s):  
Rosanna C G Smith ◽  
Patrick S Stumpf ◽  
Sonya J Ridden ◽  
Aaron Sim ◽  
Sarah Filippi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Biology ◽  
Measurement Problem ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Live Cells ◽  
Reporter Cell ◽  
Nanog Expression

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem (ES) cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, since temporal changes in intracellular protein abundances cannot be measured directly in live cells, these fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systemically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight a fundamental measurement problem in cell biology.

scholarly journals Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

2019 ◽  
Vol 317 (4) ◽  
pp. C725-C736
Author(s):  
Gurbind Singh ◽  
Divya Sridharan ◽  
Mahmood Khan ◽  
Polani B. Seshagiri
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Monochromatic Light ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Medical Diagnostics ◽  
Calcium Transients ◽  
Egfp Expression ◽  
Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

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 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.

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

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.

scholarly journals Gene Targeting of Cdc42 and Cdc42GAP Affirms the Critical Involvement of Cdc42 in Filopodia Induction, Directed Migration, and Proliferation in Primary Mouse Embryonic Fibroblasts

2006 ◽  
Vol 17 (11) ◽  
pp. 4675-4685 ◽  
Author(s):  
Linda Yang ◽  
Lei Wang ◽  
Yi Zheng
Keyword(s):  
Gene Targeting ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Cell Model ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Membrane Ruffling ◽  
Primary Mouse

Recent studies in Cdc42 knockout mouse embryonic stem (ES) cells and ES-derived fibroblastoid cell lines raise concern on a body of literature derived by dominant mutant expression approach in a variety of cell lines implicating mammalian Cdc42 as a key regulator of filopodia induction, directional migration and cell cycle progression. To resolve the physiological function of mammalian Cdc42, we have characterized the Cdc42−/− and Cdc42GAP−/− primary mouse embryonic fibroblasts (MEFs) produced by gene targeting as the Cdc42 loss- or gain-of-activity cell model. The Cdc42−/− cells were defective in filopodia formation stimulated by bradykinin and in dorsal membrane ruffling stimulated by PDGF, whereas the Cdc42GAP−/− cells displayed spontaneous filopodia. The Cdc42 loss- or gain-of-activity cells were defective in adhesion to fibronectin, wound-healing, polarity establishment, and migration toward a serum gradient. These defects were associated with deficiencies of PAK1, GSK3β, myosin light chain, and FAK phosphorylation. Furthermore, Cdc42−/− cells were defective in G1/S-phase transition and survival, correlating with deficient NF-κB transcription and defective JNK, p70 S6K, and ERK1/2 activation. These results demonstrate a different requirement of Cdc42 activity in primary MEFs from ES or ES-derived clonal fibroblastoid cells and suggest that Cdc42 plays cell-type–specific signaling roles.

Genetic Reporter Cell Lines: Tools for Stem Cell Biology and Drug Discovery

2015 ◽  
pp. 251-260
Author(s):  
Cameron P.J. Hunt ◽  
Bradley Watmuff ◽  
Brigham J. Hartley ◽  
Colin W. Pouton ◽  
John M. Haynes
Keyword(s):  
Stem Cell ◽  
Drug Discovery ◽  
Cell Lines ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Reporter Cell

290 A CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINE DERIVED FROM A SINGLE BLASTOMERE

2008 ◽  
Vol 20 (1) ◽  
pp. 224
Author(s):  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
K. Wakimoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Stage ◽  
Vitro Assay ◽  
Single Blastomere ◽  
Cell Colony

The establishment of most embryonic stem (ES) cell lines requires the destruction of embryos. Some ES cell lines in mice and humans are currently derived from a single blastomere, so that remaining blastomeres can still develop into fetuses. However, the procedures currently in use for establishing these lines are very complicated, and other ES cell lines from the same species are needed (Chung et al. 2006 Nature 439, 216–219; Klimanskaya et al. 2006 Nature 444, 481–485). The objective of this study was to devise a method simpler than those previously described for establishing ES cell lines from a single blastomere in the cynomolgus monkey. Controlled ovarian stimulation and oocyte recovery have been described previously by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI), and then cultured at 38�C in 5% CO2, 5% O2 for 2 days. The zona pellucida of 4- to 5-cell-stage embryos was disrupted using acidic Tyrode's solution, and individual blastomeres were separated from the denuded embryos using trypsin. These blastomeres were cultured on mitomycin-C-treated mouse embryonic fibroblasts and ES medium containing adrenocorticotropic hormone (ACTH) (Ogawa et al. 2004 Genes to Cells 9, 471–477). After the formation of initial outgrowths, half of the medium was changed every other day until the outgrowths reached approximately 100 cells. Passage of putative monkey ES cells was performed by mechanical dispersion of the colonies and transfer to fresh feeders every 3–4 days until there were enough cells for enzymatic dispersion. One stable ES cell line was obtained from two 4- or 5-cell-stage embryos using ES medium containing ACTH. The morphology of this ES cell colony was consistent with the monkey ES cell colony previous described by Suemori et al. (2001 Dev. Dynamics 222, 273–279). The ES cell line was passaged more than 17 times, and the morphology of the ES cell colony did not differ between the first and seventeenth passages. The ES cells showed normal karyotype and retained pluripotency markers for primate ES cells including octamer-binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, and TRA-1-81. We are presently confirming whether this ES cell line possesses potencies to differentiate in all three embryonic germ layers using both an in vitro assay and teratoma formation. Here we showed that cynomolgus monkey ES cells can be derived from a single blastomere, without co-culturing another ES cell line, as has been done in previous studies on mice and humans. This method allows the establishment of ES cell lines from a single blastomere, leaving the other blastomeres available for embryo transfer. Thus, the method described here is simpler than previously described methods and alleviates some ethical concerns.

287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES

2008 ◽  
Vol 20 (1) ◽  
pp. 223 ◽  
Author(s):  
T. Lonergan ◽  
A. Harvey ◽  
J. Zhao ◽  
B. Bavister ◽  
C. Brenner
Keyword(s):  
Cell Lines ◽  
Complex I ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Mouse Fibroblast ◽  
Es Cell ◽  
Cell Content ◽  
Stem Cell Lines

The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.

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