33 research outputs found
Defined conditions for propagation and manipulation of mouse embryonic stem cells.
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.The funding statement is uploaded separately from the manuscript but the authors acknowledged Wellcome Trust, BBSRC and MRC. Austin Smith is an MRC Professor
Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.
The transcription factors (TFs) Nanog and Esrrb playĀ important roles in embryonic stem cells (ESCs) andĀ during primordial germ-cell (PGC) development. Esrrb is a positively regulated direct target of NANOG in ESCs that can substitute qualitatively for Nanog function in ESCs. Whether this functional substitution extends to the germline is unknown. Here, we show that germline deletion of Nanog reduces PGC numbers 5-fold at midgestation. Despite this quantitative depletion, Nanog-null PGCs can complete germline development in contrast to previous findings. PGC-like cell (PGCLC) differentiation of Nanog-null ESCs is also impaired, with Nanog-null PGCLCs showing decreased proliferation and increased apoptosis. However, induced expression of Esrrb restores PGCLC numbers as efficiently as Nanog. These effects are recapitulated inĀ vivo: knockin of Esrrb to Nanog restores PGC numbers to wild-type levels and results in fertile adult mice. These findings demonstrate that Esrrb can replace Nanog function in germ cells
A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development.
Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.W.C.C.T is supported by Croucher Foundation and Cambridge Trust. P.F.C.is a Wellcome Trust Senior Fellow in Clinical Science (101876/Z/13/Z), and a UK NIHR Senior Investigator with additional support from the Wellcome Trust Centre for Mitochondrial Research (096919Z/11/Z). M.A.S. is supported by HFSP and Wellcome Trust Investigator Award.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.cell.2015.04.05
Thermal history of the plasma and high-frequency gravitons
Possible deviations from a radiation-dominated evolution, occurring prior the
synthesis of light nuclei, impacted on the spectral energy density of
high-frequency gravitons. For a systematic scrutiny of this situation, the
CDM paradigm must be complemented by (at least two) physical
parameters describing, respectively, a threshold frequency and a slope. The
supplementary frequency scale sets the lower border of a high-frequency domain
where the spectral energy grows with a slope which depends, predominantly, upon
the total sound speed of the plasma right after inflation. While the infra-red
region of the graviton energy spectrum is nearly scale-invariant, the expected
signals for typical frequencies larger than 0.01 nHz are hereby analyzed in a
model-independent framework by requiring that the total sound speed of the
post-inflationary plasma be smaller than the speed of light. Current (e.g.
low-frequency) upper limits on the tensor power spectra (determined from the
combined analysis of the three large-scale data sets) are shown to be
compatible with a detectable signal in the frequency range of wide-band
interferometers. In the present context, the scrutiny of the early evolution of
the sound speed of the plasma can then be mapped onto a reliable strategy of
parameter extraction including not only the well established cosmological
observables but also the forthcoming data from wide band interferometers.Comment: 47 pages, 31 included figures, to appear in Classical and Quantum
Gravit
Stochastic backgrounds of relic gravitons: a theoretical appraisal
Stochastic backgrounds or relic gravitons, if ever detected, will constitute
a prima facie evidence of physical processes taking place during the earliest
stages of the evolution of the plasma. The essentials of the stochastic
backgrounds of relic gravitons are hereby introduced and reviewed. The pivotal
observables customarily employed to infer the properties of the relic gravitons
are discussed both in the framework of the CDM paradigm as well as in
neighboring contexts. The complementarity between experiments measuring the
polarization of the Cosmic Microwave Background (such as, for instance, WMAP,
Capmap, Quad, Cbi, just to mention a few) and wide band interferometers (e.g.
Virgo, Ligo, Geo, Tama) is emphasized. While the analysis of the microwave sky
strongly constrains the low-frequency tail of the relic graviton spectrum,
wide-band detectors are sensitive to much higher frequencies where the spectral
energy density depends chiefly upon the (poorly known) rate of
post-inflationary expansion.Comment: 94 pages, 32 figure
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Defined conditions for propagation and manipulation of mouse embryonic stem cells.
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.The funding statement is uploaded separately from the manuscript but the authors acknowledged Wellcome Trust, BBSRC and MRC. Austin Smith is an MRC Professor
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All models are wrong, but some are useful:Establishing standards for stem cell-based embryo models
Detailed studies of the embryo allow an increasingly mechanistic understanding of development, which has proved of profound relevance to human disease. The last decade has seen inĀ vitro cultured stem cell-based models of embryo development flourish, which provide an alternative to the embryo for accessible experimentation. However, the usefulness of any stem cell-based embryo model will be determined by how accurately it reflects inĀ vivo embryonic development, and/or the extent to which it facilitates new discoveries. Stringent benchmarking of embryo models is thus an important consideration for this growing field. Here we provide an overview of means to evaluate both the properties of stem cells, the building blocks of most embryo models, as well as the usefulness of current and future inĀ vitro embryo models
Defined conditions for propagation and manipulation of mouse embryonic stem cells
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.ISSN:0950-1991ISSN:1477-912
Eggs sense high-fat diet
Maternal high-fat diet has a negative impact on fertilityāincluding an apparent direct effect on early development. In this issue, a new study connects this phenotype to depletion of Stella protein in oocytes, demonstrating environmental regulation of a maternal-effect gene
Synergistic Mechanisms of DNA Demethylation during Transition to Ground-State Pluripotency
SummaryPluripotent stem cells (PSCs) occupy a spectrum of reversible molecular states ranging from a naive ground-state in 2i, to metastable embryonic stem cells (ESCs) in serum, to lineage-primed epiblast stem cells (EpiSCs). To investigate the role of DNA methylation (5mC) across distinct pluripotent states, we mapped genome-wide 5mC and 5-hydroxymethycytosine (5hmC) in multiple PSCs. Ground-state ESCs exhibit an altered distribution of 5mC and 5hmC at regulatory elements and dramatically lower absolute levels relative to ESCs in serum. By contrast, EpiSCs exhibit increased promoter 5mC coupled with reduced 5hmC, which contributes to their developmental restriction. Switch to 2i triggers rapid onset of both the ground-state gene expression program and global DNA demethylation. Mechanistically, repression of de novo methylases by PRDM14 drives DNA demethylation at slow kinetics, whereas TET1/TET2-mediated 5hmC conversion enhances both the rate and extent of hypomethylation. These processes thus act synergistically during transition to ground-state pluripotency to promote a robust hypomethylated state