scholarly journals Endogenous epitope-tagging of Tet1, Tet2 and Tet3 identifies TET2 as a naïve pluripotency marker

2019 ◽  
Vol 2 (5) ◽  
pp. e201900516 ◽  
Author(s):  
Raphaël Pantier ◽  
Tülin Tatar ◽  
Douglas Colby ◽  
Ian Chambers
Keyword(s):  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Culture Conditions ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Pluripotency Marker ◽  
Tet Protein ◽  
Cell Culture Conditions ◽  
Facs Sorting ◽  
Pluripotency Genes

Tet1, Tet2, and Tet3 encode DNA demethylases that play critical roles during stem cell differentiation and reprogramming to pluripotency. Although all three genes are transcribed in pluripotent cells, little is known about the expression of the corresponding proteins. Here, we tagged all the endogenous Tet family alleles using CRISPR/Cas9, and characterised TET protein expression in distinct pluripotent cell culture conditions. Whereas TET1 is abundantly expressed in both naïve and primed pluripotent cells, TET2 expression is restricted to the naïve state. Moreover, TET2 is expressed heterogeneously in embryonic stem cells (ESCs) cultured in serum/leukemia inhibitory factor, with expression correlating with naïve pluripotency markers. FACS-sorting of ESCs carrying a Tet2Flag-IRES-EGFP reporter demonstrated that TET2-negative cells have lost the ability to form undifferentiated ESC colonies. We further show that TET2 binds to the transcription factor NANOG. We hypothesize that TET2 and NANOG co-localise on chromatin to regulate enhancers associated with naïve pluripotency genes.

scholarly journals Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

2016 ◽  
Vol 7 (2) ◽  
pp. 177-191 ◽  
Author(s):  
Javier Martin Gonzalez ◽  
Sophie M. Morgani ◽  
Robert A. Bone ◽  
Kasper Bonderup ◽  
Sahar Abelchian ◽  
...  
Keyword(s):  
Cell Culture ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Developmental Stages ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Stem Cell Culture ◽  
Cell Culture Conditions

scholarly journals Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Halima Albalushi ◽  
Magdalena Kurek ◽  
Leif Karlsson ◽  
Luise Landreh ◽  
Kristín Rós Kjartansdóttir ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Cell Types ◽  
Culture Conditions ◽  
Pluripotency Marker ◽  
Human Foreskin Fibroblasts ◽  
Pluripotency Gene ◽  
Hes Cells ◽  
Cells Cultured

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.

scholarly journals Developmental Activation of the Rb–E2F Pathway and Establishment of Cell Cycle-regulated Cyclin-dependent Kinase Activity during Embryonic Stem Cell Differentiation

2005 ◽  
Vol 16 (4) ◽  
pp. 2018-2027 ◽  
Author(s):  
Josephine White ◽  
Elaine Stead ◽  
Renate Faast ◽  
Simon Conn ◽  
Peter Cartwright ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Target Genes ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation ◽  
Cyclin Dependent Kinase ◽  
Pluripotent Cells ◽  
Cyclin E1 ◽  
Cdk2 Activity ◽  
Cycle Dependent

To understand cell cycle control mechanisms in early development and how they change during differentiation, we used embryonic stem cells to model embryonic events. Our results demonstrate that as pluripotent cells differentiate, the length of G1 phase increases substantially. At the molecular level, this is associated with a significant change in the size of active cyclin-dependent kinase (Cdk) complexes, the establishment of cell cycle-regulated Cdk2 activity and the activation of a functional Rb–E2F pathway. The switch from constitutive to cell cycle-dependent Cdk2 activity coincides with temporal changes in cyclin A2 and E1 protein levels during the cell cycle. Transcriptional mechanisms underpin the down-regulation of cyclin levels and the establishment of their periodicity during differentiation. As pluripotent cells differentiate and pRb/p107 kinase activities become cell cycle dependent, the E2F–pRb pathway is activated and imposes cell cycle-regulated transcriptional control on E2F target genes, such as cyclin E1. These results suggest the existence of a feedback loop where Cdk2 controls its own activity through regulation of cyclin E1 transcription. Changes in rates of cell division, cell cycle structure and the establishment of cell cycle-regulated Cdk2 activity can therefore be explained by activation of the E2F–pRb pathway.

Germ-cell culture conditions facilitate the production of mouse embryonic stem cells

2014 ◽  
Vol 81 (9) ◽  
pp. 794-804
Author(s):  
Priscila Ramos-Ibeas ◽  
Eva Pericuesta ◽  
Raúl Fernández-González ◽  
Alfonso Gutiérrez-Adán ◽  
Miguel Ángel Ramírez
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Culture Conditions ◽  
Cell Culture Conditions

scholarly journals Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development.

2002 ◽  
Vol 115 (2) ◽  
pp. 329-339 ◽  
Author(s):  
T. A. Pelton ◽  
S. Sharma ◽  
T. C. Schulz ◽  
J. Rathjen ◽  
P. D. Rathjen
Keyword(s):  
Cell Mass ◽  
Embryonic Stem ◽  
Indirect Evidence ◽  
Cell Populations ◽  
Molecular Evidence ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Cell Progression

Formation and differentiation of a pluripotent cell population is central to mammalian development, and the isolation, identification and manipulation of human pluripotent cells is predicted to be of therapeutic use. Within the early mammalian embryo, two distinct populations of pluripotent cells have been described: the inner cell mass (ICM), which differentiates to form a second pluripotent cell populations, the primitive ectoderm. Indirect evidence suggests the existence of temporally distinct intermediate pluripotent cell populations as primitive ectoderm is formed. We coupled an in vitro model of primitive ectoderm formation (the transition of embryonic stem cells to early primitive ectoderm-like (EPL) cells) with ddPCR-based techniques to identify three novel genes, Psc1, CRTR-1 and PRCE, that were expressed differently during pluripotent cell progression. Detailed mapping of these genes with Oct4, Rex1 and Fgf5 on pregastrulation embryos provided the first molecular evidence for the existence of successive, temporally distinct pluripotent cell populations in the embryo between the ICM and primitive ectoderm. No evidence was found for spatial heterogeneity within the Oct4+ pool. The transition between populations correlated with morphological or developmental alterations in pluripotent cells in vivo. Genes that are temporally expressed during pluripotent cell progression may provide an opportunity for molecular discrimination of pluripotent cells at different stages of maturation in vivo and an understanding of the cellular origins and properties of pluripotent cell lines isolated from diverse sources. Furthermore, the strong correlation of gene expression demonstrated between EPL cell formation in vitro and primitive ectoderm formation in vivo validates EPL cells as a model for primitive ectoderm, thereby providing a model system for the investigation of pluripotent differentiation and an opportunity for directed differentiation of pluripotent cells to therapeutically useful cell populations.

Lectin Binding Studies on RNA Tumor Virus-Infected Cells

1975 ◽  
Vol 33 ◽  
pp. 326-327
Author(s):  
D. C. Hixson
Keyword(s):  
Binding Sites ◽  
Lectin Binding ◽  
Culture Conditions ◽  
3T3 Cells ◽  
Binding Studies ◽  
Con A ◽  
Microscopic Studies ◽  
Tumor Virus ◽  
Infected Cells ◽  
Cell Culture Conditions

The abilities of plant lectins to preferentially agglutinate malignant cells and to bind to specific monosaccharide or oligosaccharide sequences of glycoproteins and glycolipids make them a new and important biochemical probe for investigating alterations in plasma membrane structure which may result from malignant transformation. Electron and light microscopic studies have demonstrated clustered binding sites on surfaces of SV40-infected or tryp- sinized 3T3 cells when labeled with concanavalin A (con A). No clustering of con A binding sites was observed in normal 3T3 cells. It has been proposed that topological rearrangement of lectin binding sites into clusters enables con A to agglutinate SV40-infected or trypsinized 3T3 cells (1). However, observations by other investigators have not been consistent with this proposal (2) perhaps due to differences in reagents used, cell culture conditions, or labeling techniques. The present work was undertaken to study the lectin binding properties of normal and RNA tumor virus-infected cells and their associated viruses using lectins and ferritin-conjugated lectins of five different specificities.

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