scholarly journals DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

2020 ◽  
Vol 6 (35) ◽  
pp. eabb5820 ◽  
Author(s):  
Zhiming Li ◽  
Xu Hua ◽  
Albert Serra-Cardona ◽  
Xiaowei Xu ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endogenous Retroviruses ◽  
Histone Binding ◽  
Dna Polymerase Α ◽  
Mouse Es Cells ◽  
Dna Strands ◽  
Histone Deposition

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

Study of the C60 Fullerene on Differentiation of Mouse Embryonic Stem Cells

2012 ◽  
Vol 529-530 ◽  
pp. 385-390
Author(s):  
Koichi Imai ◽  
Fumio Watari ◽  
Kazuaki Nakamura ◽  
Akito Tanoue
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Experimental Method ◽  
Es Cells ◽  
Embryonic Stem ◽  
Future Generations ◽  
C60 Fullerene ◽  
Biological Safety ◽  
Mouse Es Cells

The risks of nanomaterials for future generations should be elucidated. Thus, it is important to establish an experimental method to accurately examine embryotoxicity. We have conducted anin vitroembryotoxicity test with mouse ES cells to examine the embryotoxicities of various nanomaterials. In this study, the C60 fullerene did not influence the differentiation of ES-D3 cells and "non embryotoxicity". In the future, the biological safety should be comprehensively examined by improving dispersion in medium.

Role of the phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1522-1525 ◽  
Author(s):  
K. Takahashi ◽  
M. Murakami ◽  
S. Yamanaka
Keyword(s):  
Es Cells ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Pi3k Pathway ◽  
Leukaemia Inhibitory Factor ◽  
Mouse Es Cells ◽  
Ras Family ◽  
Phosphoinositide 3 Kinase ◽  
Kinase Pathway

Mouse ES (embryonic stem) cells maintain pluripotency with robust proliferation in vitro. ES cells share some similarities with cancer cells, such as anchorage-independent growth, loss of contact inhibition and tumour formation. After differentiation, ES cells lose pluripotency and tumorigenicity. Recent studies showed that the PI3K (phosphoinositide 3-kinase) pathway is important for proliferation, survival and maintenance of pluripotency in ES cells. The PI3K pathway is activated by growth factors and cytokines including insulin and leukaemia inhibitory factor. In addition to these exogenous factors, the PI3K pathway is endogenously activated by the constitutively active Ras family protein ERas (ES cell-expressed Ras). The PI3K pathway utilizes multiple downstream effectors including mTOR (mammalian target of rapamycin), which we have shown to be essential for proliferation in mouse ES cells and early embryos.

220 NONHUMAN PRIMATE EMBRYONIC STEM CELLS SIMILAR TO THE BIOLOGICAL PROPERTIES OF MOUSE EMBRYONIC STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 222
Author(s):  
A. Kusanagi ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Nonhuman Primate ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Biological Properties ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Mouse Es Cell

Human and mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts and human ES cells were long thought to be equivalent to mouse ES cells, despite clear morphological difference and different signalling pathways to maintain their pluripotency between these two ES cell types. Mouse ES cells depend on leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signalling, whereas their human counterparts rely on basic fibroblast growth factor (bFGF) and activin A signalling. The biggest difference of two ES cells is the ability of chimera formation and mouse ES cells can contribute chimera but primate ES cells fails to do that. Monkey ES cells in primates only can be tested for chimera formation in vivo due to the ethical issue and cynomolgus monkey is the most common nonhuman primate to be used for the safety study of drug discoveries. The objective of this study was to develop novel cynomolgus monkey ES cells that have similar biological properties with mouse ES cell and our ultimate goal is to establish germline competent nonhuman primate ES cells. Ovarian stimulation and oocyte collection were carried out for the derivation of ES cells as previously described by Torii et al. Briefly, GnRH (0.9 mg/head) was administered to cynomolgus monkey and two weeks later, a micro infusion pump (iPRECIO™, Primetech Corp) contains FSH was implanted subcutaneously. Follicular aspiration was then performed 40 h after hCG injection and metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Cynomolgus monkey ES cells were then established under mouse ES cell conditions such as LIF/STAT signalling and a dome tree-dimensional (3D) morphology nonhuman primate ES cells were selected. On the other hands, ES cells that were established with the presence of basic FGF showed conventional layer-type morphology. Dome-type ES cells express pluripotent transcriptional factors such as Oct-3/4, Nonog and Sox2 as same as layer-type ES cells and both ES lines were capable of multilineage differentiations in vitro after embryoid body formation. Dome-type nonhuman ES cells can also form teratomas and differentiated into all three germ layers when grafted into immunodeficiency mice. For fluorescent gene delivery to nonhuman primate ES cells, feeder-free condition was applied and CAG-GFP vector was transfected into ES cells using Neon electroporation system (Invitrogen Inc.) for the tracing ES cells in the transplantation study. In this study, we have established dome-type ES cell lines that similar to mouse ES cells in morphology and signalling pathway. Dome-type nonhuman primate ES cells express pluripotent gene markers and prove their pluripotency both of in vitro and in vivo, in addition, these modifications would be important to create germline competent ES cells.

scholarly journals Conserved and divergent features of DNA methylation in embryonic stem cell-derived neurons

2020 ◽  
Author(s):  
Sally Martin ◽  
Daniel Poppe ◽  
Nelly Olova ◽  
Conor O’Leary ◽  
Elena Ivanova ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Nuclear Lamina ◽  
Neuronal Maturation ◽  
Neuronal Marker ◽  
Mouse Es Cells ◽  
Primary Mouse

AbstractDNA methylation functions in genome regulation and is implicated in neuronal maturation. Early post-natal accumulation of atypical non-CG methylation (mCH) occurs in neurons of mice and humans, but its precise function remains unknown. Here we investigate mCH deposition in neurons derived from mouse ES-cells in vitro and in cultured primary mouse neurons. We find that both acquire comparable levels of mCH over a similar period as in vivo. In vitro mCH deposition occurs concurrently with a transient increase in Dnmt3a expression, is preceded by expression of the post-mitotic neuronal marker Rbfox3 (NeuN) and is enriched at the nuclear lamina. Despite these similarities, whole genome bisulfite sequencing reveals that mCH patterning in mESC-derived neurons partially differs from in vivo. mESC-derived neurons therefore represent a valuable model system for analyzing the mechanisms and functional consequences of correct and aberrantly deposited CG and non-CG methylation in neuronal maturation.

scholarly journals Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway

2012 ◽  
Vol 287 (42) ◽  
pp. 35599-35611 ◽  
Author(s):  
Wen-Jie Wei ◽  
Hai-Ying Sun ◽  
Kai Yiu Ting ◽  
Li-He Zhang ◽  
Hon-Cheung Lee ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Embryoid Body ◽  
Troponin T ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Adp Ribosyl Cyclase

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca2+ in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca2+ signaling pathway antagonizes the CM differentiation of mouse ES cells.

Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy

2005 ◽  
Vol 85 (2) ◽  
pp. 635-678 ◽  
Author(s):  
Anna M. Wobus ◽  
Kenneth R. Boheler
Keyword(s):  
Developmental Biology ◽  
Cell Lines ◽  
Tissue Regeneration ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Human Es Cells

Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.

Transcriptional heterogeneity in mouse embryonic stem cells

2009 ◽  
Vol 21 (1) ◽  
pp. 67 ◽  
Author(s):  
Tetsuya S. Tanaka
Keyword(s):  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
The Body ◽  
Cell Subpopulation ◽  
Es Cell ◽  
Differentiation Potential ◽  
Mouse Es Cells

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.

Studies of in vitro differentiation with embryonic stem cells

1994 ◽  
Vol 6 (5) ◽  
pp. 543 ◽  
Author(s):  
RA Pedersen
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Models ◽  
Embryonic Cell ◽  
Mammalian Embryo ◽  
Mouse Es Cells ◽  
Molecular Events ◽  
Differentiation And Proliferation

Embryonic stem (ES) cells were first cultured from mouse embryos little more than a decade ago, yet they are now widely used in transgenic studies which are revolutionizing mammalian genetics. Although drawing less attention, in vitro studies of mouse ES cells have also contributed widely to the understanding of mechanisms of embryonic cell differentiation and proliferation. This review focusses on the application of ES cells as in vitro models for cellular and molecular events in the early mammalian embryo. Future studies with cultured ES cells of mouse and other species should provide insights into the factors regulating the differentiation of intermediate stem cells and terminal cells for the various embryonic lineages, thus contributing profoundly to the understanding of mammalian embryogenesis as well as providing cells for therapeutic applications.

scholarly journals Generation of Aggregates of Mouse ES Cells that Show Symmetry Breaking, Polarisation and Emergent Collective Behaviour in vitro.

2014 ◽  
Author(s):  
Peter Baillie-Johnson ◽  
Susanne C van den Brink ◽  
Tina Balayo ◽  
David A Turner ◽  
Alfonso Martinez Arias
Keyword(s):  
Symmetry Breaking ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basal Medium ◽  
Cell Types ◽  
Mouse Es Cells ◽  
Cellular Decision Making ◽  
Adherent Culture ◽  
Early Developmental

Dissociated mouse embryonic stem (ES) cells were cultured to form aggregates in small volumes of basal medium in U-bottomed, non tissue-culture-treated 96-well plates and subsequently maintained in suspension culture. After growth for 48 hours, the aggregates are competent to respond to ubiquitous experimental signals which result in their symmetry-breaking and generation of defined polarised structures by 96 hours. It is envisaged that this system can be applied both to the study of early developmental events and more broadly to the processes of self-organisation and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo but unobtainable from conventional adherent culture.

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