Publications by authors named "Harry G Leitch"

27 Publications

  • Page 1 of 1

All models are wrong, but some are useful: Establishing standards for stem cell-based embryo models.

Stem Cell Reports 2021 May;16(5):1117-1141

MRC London Institute of Medical Sciences, London, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London W12 0NN, UK; Centre for Paediatrics and Child Health, Faculty of Medicine, Imperial College London, London W2 1PG, UK.

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 in 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 in 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 in vitro embryo models.
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http://dx.doi.org/10.1016/j.stemcr.2021.03.019DOI Listing
May 2021

Reconstitution of the oocyte transcriptional network with transcription factors.

Nature 2021 01 16;589(7841):264-269. Epub 2020 Dec 16.

Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.

During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.
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http://dx.doi.org/10.1038/s41586-020-3027-9DOI Listing
January 2021

Mouse Primordial Germ Cells: In Vitro Culture and Conversion to Pluripotent Stem Cell Lines.

Methods Mol Biol 2021 ;2214:59-73

MRC London Institute of Medical Sciences (LMS), London, UK.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes. Despite decades of research, in vitro culture of PGCs remains a major challenge and has previously relied on undefined components such as serum and feeders. Notably, PGCs cultured for extended periods do not maintain their lineage identity but instead undergo conversion to form pluripotent stem cell lines called embryonic germ (EG) cells in response to LIF/STAT3 signaling. Here we report both established and new methodologies to derive EG cells, in a range of different conditions. We show that basic fibroblast growth factor is not required for EG cell conversion. We detail the steps taken in our laboratory to systematically remove complex components and establish a fully defined protocol that allows efficient conversion of isolated PGCs to pluripotent EG cells. In addition, we demonstrate that PGCs can adhere and proliferate in culture without the support of feeder cells or serum. This may well suggest novel approaches to establishing short-term culture of PGCs in defined conditions.
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http://dx.doi.org/10.1007/978-1-0716-0958-3_5DOI Listing
March 2021

Defined conditions for propagation and manipulation of mouse embryonic stem cells.

Development 2019 03 26;146(6). Epub 2019 Mar 26.

Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Gleeson Building, Tennis Court Road, Cambridge CB2 1QR, UK

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.
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http://dx.doi.org/10.1242/dev.173146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6451320PMC
March 2019

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA.

Genome Res 2018 12 7;28(12):1943-1956. Epub 2018 Nov 7.

Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London W12 0NN, United Kingdom.

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5' ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of the rules of transcription initiation, led to discovery of new core promoter sequence features, and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 µg), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5' ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. This dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods, by generating a complex, high-quality library from mouse embryonic day 11.5 primordial germ cells.
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http://dx.doi.org/10.1101/gr.235937.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280763PMC
December 2018

Publisher Correction: Eggs sense high-fat diet.

Nat Genet 2018 08;50(8):1196

MRC London Institute of Medical Sciences (LMS), London, UK.

In the version of this article originally published, a box was misplaced in Fig. 1. The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41588-018-0137-5DOI Listing
August 2018

Epigenetic reprogramming enables the transition from primordial germ cell to gonocyte.

Nature 2018 03 7;555(7696):392-396. Epub 2018 Mar 7.

MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN, UK.

Gametes are highly specialized cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mice, germ cells are first specified in the developing embryo around embryonic day (E) 6.25 as primordial germ cells (PGCs). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming around E10.5-E11.5, including genome-wide loss of 5-methylcytosine. The underlying molecular mechanisms of this process have remained unclear, leading to our inability to recapitulate this step of germline development in vitro. Here we show, using an integrative approach, that this complex reprogramming process involves coordinated interplay among promoter sequence characteristics, DNA (de)methylation, the polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of TET1 to enable the activation of a critical set of germline reprogramming-responsive genes involved in gamete generation and meiosis. Our results also reveal an unexpected role for TET1 in maintaining but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will help to guide attempts to recapitulate complete gametogenesis in vitro.
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http://dx.doi.org/10.1038/nature25964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856367PMC
March 2018

Eggs sense high-fat diet.

Nat Genet 2018 03;50(3):318-319

MRC London Institute of Medical Sciences (LMS), London, UK.

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http://dx.doi.org/10.1038/s41588-018-0068-1DOI Listing
March 2018

Esrrb Complementation Rescues Development of Nanog-Null Germ Cells.

Cell Rep 2018 01;22(2):332-339

MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland. Electronic address:

The transcription factors (TFs) Nanog and Esrrb play important roles in embryonic stem cells (ESCs) and 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 in 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.
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http://dx.doi.org/10.1016/j.celrep.2017.12.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775501PMC
January 2018

DNA (De)Methylation: The Passive Route to Naïvety?

Trends Genet 2016 10 17;32(10):592-595. Epub 2016 Aug 17.

MRC Clinical Sciences Centre, Du Cane Road, London W12 0NN, UK; Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, UK. Electronic address:

Mouse pluripotent embryonic stem (ES) cells can exist in distinct yet interchangeable epigenetic states dictated by their culture environment. Previous reports have shown that naïve pluripotent cells grown in the presence of 2i are characterised by global DNA hypomethylation and changes in the abundance and distribution of histone modifications. New research provides insights regarding how this might be achieved.
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http://dx.doi.org/10.1016/j.tig.2016.08.005DOI Listing
October 2016

Hallmarks of pluripotency.

Nature 2015 Sep;525(7570):469-78

Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children's Hospital Boston, and Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.
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http://dx.doi.org/10.1038/nature15515DOI Listing
September 2015

A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development.

Cell 2015 Jun;161(6):1453-67

Wellcome Trust Cancer Research UK Gurdon Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 1QN, UK; Department of Physiology, Development and Neuroscience, Downing Street, University of Cambridge, Cambridge CB2 3EG, UK; Wellcome Trust-Medical Research Council Stem Cell Institute, Tennis Court Road, University of Cambridge, Cambridge CB2 3EG, UK. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cell.2015.04.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459712PMC
June 2015

Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency.

Stem Cell Reports 2013 17;1(6):518-31. Epub 2013 Dec 17.

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK ; Wellcome Trust/MRC Stem Cell Institute, University of Cambridge, Cambridge CB2 1QR, UK ; Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK.

Pluripotent 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.
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http://dx.doi.org/10.1016/j.stemcr.2013.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871394PMC
May 2015

On the fate of primordial germ cells injected into early mouse embryos.

Dev Biol 2014 Jan 20;385(2):155-9. Epub 2013 Nov 20.

Department of Genetics and Development, Columbia University Medical Center, New York, NY, USA. Electronic address:

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells - pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.
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http://dx.doi.org/10.1016/j.ydbio.2013.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928994PMC
January 2014

Rebuilding pluripotency from primordial germ cells.

Stem Cell Reports 2013 4;1(1):66-78. Epub 2013 Jun 4.

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK ; Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.

Mammalian primordial germ cells (PGCs) are unipotent progenitors of the gametes. Nonetheless, they can give rise directly to pluripotent stem cells in vitro or during teratocarcinogenesis. This conversion is inconsistent, however, and has been difficult to study. Here, we delineate requirements for efficient resetting of pluripotency in culture. We demonstrate that in defined conditions, routinely 20% of PGCs become EG cells. Conversion can occur from the earliest specified PGCs. The entire process can be tracked from single cells. It is driven by leukemia inhibitory factor (LIF) and the downstream transcription factor STAT3. In contrast, LIF signaling is not required during germ cell ontogeny. We surmise that ectopic LIF/STAT3 stimulation reconstructs latent pluripotency and self-renewal. Notably, STAT3 targets are significantly upregulated in germ cell tumors, suggesting that dysregulation of this pathway may underlie teratocarcinogenesis. These findings demonstrate that EG cell formation is a robust experimental system for exploring mechanisms involved in reprogramming and cancer.
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http://dx.doi.org/10.1016/j.stemcr.2013.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757743PMC
May 2015

The mammalian germline as a pluripotency cycle.

Development 2013 Jun;140(12):2495-501

Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK.

Naive pluripotency refers to the capacity of single cells in regulative embryos to engender all somatic and germline cell types. Only germ cells - conventionally considered to be unipotent - can naturally re-acquire pluripotency, by cycling through fertilisation. Furthermore, primordial germ cells express, and appear to be functionally dependent upon, transcription factors that characterise the pluripotent state. We hypothesise that germ cells require pluripotency factors to control a de-restricted epigenome. Consequently, they harbour latent potential, as manifested in teratocarcinogenesis or direct conversion into pluripotent stem cells in vitro. Thus, we suggest that there exists an unbroken cycle of pluripotency, naive in the early epiblast and latent in the germline, that is sustained by a shared transcription factor network.
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http://dx.doi.org/10.1242/dev.091603DOI Listing
June 2013

The impact of culture on epigenetic properties of pluripotent stem cells and pre-implantation embryos.

Biochem Soc Trans 2013 Jun;41(3):711-9

Medical Research Council Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, U.K.

Cultured pluripotent stem cells hold great promise for regenerative medicine. Considerable efforts have been invested into the refinement and definition of improved culture systems that sustain self-renewal and avoid differentiation of pluripotent cells in vitro. Recent studies have, however, found that the choice of culture condition has a significant impact on epigenetic profiles of cultured pluripotent cells. Mouse and human ESCs (embryonic stem cells) show substantial epigenetic differences that are dependent on the culture condition, including global changes to DNA methylation and histone modifications and, in female human ESCs, to the epigenetic process of X chromosome inactivation. Epigenetic perturbations have also been detected during culture of pre-implantation embryos; limited research undertaken in mouse suggests a direct effect of the in vitro environment on epigenetic processes in this system. Widespread epigenetic changes induced by the culture condition in stem cells thus emphasize the necessity for extensive research into both immediate and long-term epigenetic effects of embryo culture during assisted reproductive technologies.
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http://dx.doi.org/10.1042/BST20130049DOI Listing
June 2013

Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation.

EMBO Rep 2013 Jul 14;14(7):629-37. Epub 2013 May 14.

Wellcome Trust/Cancer Research UK Gurdon Institute, Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
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http://dx.doi.org/10.1038/embor.2013.67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3701237PMC
July 2013

Primordial germ-cell development and epigenetic reprogramming in mammals.

Curr Top Dev Biol 2013 ;104:149-87

Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, United Kingdom.

Primordial germ cells (PGCs) are the embryonic precursors of the gametes and represent the founder cells of the germline. Specification of PGCs is a critical divergent point during embryogenesis. Whereas the somatic lineages will ultimately perish, cells of the germline have the potential to form a new individual and hence progress to the next generation. It is therefore critical that the genome emerges intact and carrying the appropriate epigenetic information during its passage through the germline. To ensure this fidelity of transmission, PGC development encompasses extensive epigenetic reprogramming. The low cell numbers and relative inaccessibility of PGCs present a challenge to those seeking mechanistic understanding of the crucial developmental and epigenetic processes in this most fascinating of lineages. Here, we present an overview of PGC development in the mouse and compare this with the limited information available for other mammalian species. We believe that a comparative approach will be increasingly important to uncover the extent to which mechanisms are conserved and reveal the critical steps during PGC development in humans.
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http://dx.doi.org/10.1016/B978-0-12-416027-9.00005-XDOI Listing
March 2014

Different roles for Tet1 and Tet2 proteins in reprogramming-mediated erasure of imprints induced by EGC fusion.

Mol Cell 2013 Mar 28;49(6):1023-33. Epub 2013 Feb 28.

Lymphocyte Development Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK.

Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
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http://dx.doi.org/10.1016/j.molcel.2013.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3613797PMC
March 2013

Naive pluripotency is associated with global DNA hypomethylation.

Nat Struct Mol Biol 2013 Mar 17;20(3):311-6. Epub 2013 Feb 17.

Wellcome Trust/Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK.

Naive pluripotent embryonic stem cells (ESCs) and embryonic germ cells (EGCs) are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs, in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however, we find that EGCs and ESCs exhibit equivalent global DNA methylation levels. Inhibition of MEK and Gsk3b by 2i conditions leads to pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i, and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instills a naive pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.
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http://dx.doi.org/10.1038/nsmb.2510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591483PMC
March 2013

Culture parameters for stable expansion, genetic modification and germline transmission of rat pluripotent stem cells.

Biol Open 2012 Jan 1;1(1):58-65. Epub 2011 Nov 1.

Wellcome Trust Centre for Stem Cell Research, University of Cambridge , Tennis Court Road, Cambridge CB2 1QN , UK ; Department of Biochemistry, University of Cambridge , Tennis Court Road, Cambridge CB2 1QN , UK.

The ability of cultured pluripotent cells to contribute to the germline of chimaeric animals is essential to their utility for genetic manipulation. In the three years since rat embryonic stem (ES) cells were first reported the anticipated proliferation of genetically modified rat models from this new resource has not been realised. Culture instability, karyotypic anomalies, and strain variation are postulated to contribute to poor germline colonisation capacity. The resolution of these issues is essential to bring pluripotent cell-based genetic manipulation technology in the rat to the level of efficiency achieved in the mouse. Recent reports have described various alternative methods to maintain rat ES cells that include provision of additional small molecules and selective passaging methods. In contrast, we report that euploid, germline competent rat ES and embryonic germ (EG) cell lines can be maintained by simple adherent culture methods in defined medium supplemented with the original two inhibitors (2i) of the mitogen-activated protein kinase (ERK1/2) cascade and of glycogen synthase kinase 3, in combination with the cytokine leukaemia inhibitory factor (LIF). We demonstrate genetic modification, clonal expansion and transmission through the germline of rat ES and EG cell lines. We also describe a marked preference for full-term chimaera contribution when SD strain blastocysts are used as recipients for either DA or SD pluripotent stem cells.
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http://dx.doi.org/10.1242/bio.2011029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3507162PMC
January 2012

The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells.

Cell Stem Cell 2012 Jul;11(1):110-7

Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK.

Blimp1 (Prdm1), the key determinant of primordial germ cells (PGCs), plays a combinatorial role with Prdm14 during PGC specification from postimplantation epiblast cells. They together initiate epigenetic reprogramming in early germ cells toward an underlying pluripotent state, which is equivalent to embryonic stem cells (ESCs). Whereas Prdm14 alone can promote reprogramming and is important for the propagation of the pluripotent state, it is not known whether Blimp1 is similarly involved. By using a genetic approach, we demonstrate that Blimp1 is dispensable for the derivation and maintenance of ESCs and postimplantation epiblast stem cells (epiSCs). Notably, Blimp1 is also dispensable for reprogramming epiSCs to ESCs. Thus, although Blimp1 is obligatory for PGC specification, it is not required for the reversion of epiSCs to ESCs and for their maintenance thereafter. This study suggests that reprogramming, including that of somatic cells to ESCs, may not entail an obligatory route through a Blimp1-positive PGC-like state.
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http://dx.doi.org/10.1016/j.stem.2012.02.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391686PMC
July 2012

Embryonic germ cells from mice and rats exhibit properties consistent with a generic pluripotent ground state.

Development 2010 Jul 2;137(14):2279-87. Epub 2010 Jun 2.

Wellcome Trust Cancer Research UK Gurdon Institute, The Henry Wellcome Building of Cancer and Developmental Biology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

Mouse and rat embryonic stem cells can be sustained in defined medium by dual inhibition (2i) of the mitogen-activated protein kinase (Erk1/2) cascade and of glycogen synthase kinase 3. The inhibitors suppress differentiation and enable self-renewal of pluripotent cells that are ex vivo counterparts of naïve epiblast cells in the mature blastocyst. Pluripotent stem cell lines can also be derived from unipotent primordial germ cells via a poorly understood process of epigenetic reprogramming. These are termed embryonic germ (EG) cells to denote their distinct origin. Here we investigate whether EG cell self-renewal and derivation are supported by 2i. We report that mouse EG cells can be established with high efficiency using 2i in combination with the cytokine leukaemia inhibitory factor (LIF). Furthermore, addition of fibroblast growth factor or stem cell factor is unnecessary using 2i-LIF. The derived EG cells contribute extensively to healthy chimaeric mice, including to the germline. Using the same conditions, we describe the first derivations of EG cells from the rat. Rat EG cells express a similar marker profile to rat and mouse ES cells. They have a diploid karyotype, can be clonally expanded and genetically manipulated, and are competent for multilineage colonisation of chimaeras. These findings lend support to the postulate of a conserved molecular ground state in pluripotent rodent cells. Future research will determine the extent to which this is maintained in other mammals and whether, in some species, primordial germ cells might be a more tractable source than epiblast for the capture of naïve pluripotent stem cells.
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http://dx.doi.org/10.1242/dev.050427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2889601PMC
July 2010

Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene.

Proc Natl Acad Sci U S A 2007 Jun 11;104(25):10714-9. Epub 2007 Jun 11.

Reproductive Physiology Group, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge CB2 3EG, United Kingdom.

The G protein-coupled receptor GPR54 (AXOR12, OT7T175) is central to acquisition of reproductive competency in mammals. Peptide ligands (kisspeptins) for this receptor are encoded by the Kiss1 gene, and administration of exogenous kisspeptins stimulates hypothalamic gonadotropin-releasing hormone (GnRH) release in several species, including humans. To establish that kisspeptins are the authentic agonists of GPR54 in vivo and to determine whether these ligands have additional physiological functions we have generated mice with a targeted disruption of the Kiss1 gene. Kiss1-null mice are viable and healthy with no apparent abnormalities but fail to undergo sexual maturation. Mutant female mice do not progress through the estrous cycle, have thread-like uteri and small ovaries, and do not produce mature Graffian follicles. Mutant males have small testes, and spermatogenesis arrests mainly at the early haploid spermatid stage. Both sexes have low circulating gonadotropin (luteinizing hormone and follicle-stimulating hormone) and sex steroid (beta-estradiol or testosterone) hormone levels. Migration of GnRH neurons into the hypothalamus appears normal with appropriate axonal connections to the median eminence and total GnRH content. The hypothalamic-pituitary axis is functional in these mice as shown by robust luteinizing hormone secretion after peripheral administration of kisspeptin. The virtually identical phenotype of Gpr54- and Kiss1-null mice provides direct proof that kisspeptins are the true physiological ligand for the GPR54 receptor in vivo. Kiss1 also does not seem to play a vital role in any other physiological processes other than activation of the hypothalamic-pituitary-gonadal axis, and loss of Kiss1 cannot be overcome by compensatory mechanisms.
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http://dx.doi.org/10.1073/pnas.0704114104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1965578PMC
June 2007