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  1. Article ; Online: Induction of Human Naïve Pluripotency Using Chemical Resetting.

    Rugg-Gunn, Peter J

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2416, Page(s) 29–37

    Abstract: Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve pluripotent stem cells can be captured directly from ... ...

    Abstract Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve pluripotent stem cells can be captured directly from blastocysts but, more commonly, the cells are reprogrammed from primed cells in a process called "resetting". Several methods to achieve resetting have been described. Chemical resetting of primed cells to a naïve pluripotent state is one such method and has come to the forefront as a simple, efficient, and transgene-free method to induce naïve pluripotency. The process involves the transient application of a histone deacetylase inhibitor to initiate resetting, followed by the emergence of nascent naïve pluripotent stem cells in supportive conditions, and finally the stabilization and expansion of naïve pluripotent stem cell cultures. Here, a detailed protocol is provided for chemical resetting starting from plating primed cells until a stable culture of naïve pluripotent stem cells is established.
    MeSH term(s) Blastocyst ; Cell Differentiation ; Germ Layers ; Humans ; Pluripotent Stem Cells
    Language English
    Publishing date 2021-12-02
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1908-7_3
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  2. Article ; Online: Flow Cytometry Analysis of Cell-Surface Markers to Identify Human Naïve Pluripotent Stem Cells.

    Rugg-Gunn, Peter J

    Methods in molecular biology (Clifton, N.J.)

    2021  Volume 2416, Page(s) 257–265

    Abstract: Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell ... ...

    Abstract Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content.
    MeSH term(s) Biomarkers ; Cell Differentiation ; Flow Cytometry ; Humans ; Membrane Proteins ; Pluripotent Stem Cells
    Chemical Substances Biomarkers ; Membrane Proteins
    Language English
    Publishing date 2021-12-01
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-1908-7_16
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  3. Article ; Online: Epigenetic regulation of early human embryo development.

    Wilkinson, Amy L / Zorzan, Irene / Rugg-Gunn, Peter J

    Cell stem cell

    2023  Volume 30, Issue 12, Page(s) 1569–1584

    Abstract: Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now ... ...

    Abstract Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.
    MeSH term(s) Humans ; DNA Methylation ; Embryo, Mammalian/metabolism ; Embryonic Development/genetics ; Epigenesis, Genetic
    Language English
    Publishing date 2023-10-18
    Publishing country United States
    Document type Journal Article ; Review ; Research Support, Non-U.S. Gov't
    ZDB-ID 2375354-7
    ISSN 1875-9777 ; 1934-5909
    ISSN (online) 1875-9777
    ISSN 1934-5909
    DOI 10.1016/j.stem.2023.09.010
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  4. Article ; Online: Transcription factors make the right contacts.

    Rugg-Gunn, Peter J

    Nature cell biology

    2019  Volume 21, Issue 10, Page(s) 1173–1174

    MeSH term(s) Animals ; Chromatin Assembly and Disassembly/genetics ; Enhancer Elements, Genetic ; Kruppel-Like Transcription Factors/metabolism ; Mice ; Pluripotent Stem Cells/metabolism
    Chemical Substances GKLF protein ; Kruppel-Like Transcription Factors
    Language English
    Publishing date 2019-09-23
    Publishing country England
    Document type Journal Article
    ZDB-ID 1474722-4
    ISSN 1476-4679 ; 1465-7392
    ISSN (online) 1476-4679
    ISSN 1465-7392
    DOI 10.1038/s41556-019-0399-x
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  5. Article ; Online: Technical challenges of studying early human development.

    Rugg-Gunn, Peter J / Moris, Naomi / Tam, Patrick P L

    Development (Cambridge, England)

    2023  Volume 150, Issue 11

    Abstract: Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo ... ...

    Abstract Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.
    MeSH term(s) Humans ; Embryonic Development/genetics ; Gastrulation ; Embryo, Mammalian ; Stem Cells
    Language English
    Publishing date 2023-06-01
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 90607-4
    ISSN 1477-9129 ; 0950-1991
    ISSN (online) 1477-9129
    ISSN 0950-1991
    DOI 10.1242/dev.201797
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  6. Article ; Online: Naive pluripotent stem cells as a model for studying human developmental epigenomics: opportunities and limitations.

    Rugg-Gunn, Peter J

    Epigenomics

    2017  Volume 9, Issue 12, Page(s) 1485–1488

    MeSH term(s) DNA Methylation ; Embryonic Stem Cells/cytology ; Epigenomics ; Humans ; X Chromosome Inactivation
    Language English
    Publishing date 2017-11-06
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1750-192X
    ISSN (online) 1750-192X
    DOI 10.2217/epi-2017-0115
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  7. Article ; Online: Amniogenesis occurs in two independent waves in primates.

    Rostovskaya, Maria / Andrews, Simon / Reik, Wolf / Rugg-Gunn, Peter J

    Cell stem cell

    2022  Volume 29, Issue 5, Page(s) 744–759.e6

    Abstract: In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of amniogenesis diversified during evolution remains ... ...

    Abstract In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of amniogenesis diversified during evolution remains unknown. Unexpectedly, single-cell analysis of primate embryos uncovered two transcriptionally and temporally distinct amniogenesis waves. To study this, we employed the naive-to-primed transition of human pluripotent stem cells (hPSCs) to model peri-implantation epiblast development. Partially primed hPSCs transiently gained the ability to differentiate into cavitating epithelium that transcriptionally and morphologically matched the early amnion, whereas fully primed hPSCs produced cells resembling the late amnion instead, thus recapitulating the two independent differentiation waves. The early wave follows a trophectoderm-like pathway and encompasses cavitation, whereas the late wave resembles an ectoderm-like route during gastrulation. The discovery of two independent waves explains how amniogenesis through cavitation could emerge during evolution via duplication of the pre-existing trophectoderm program.
    MeSH term(s) Animals ; Cell Differentiation ; Ectoderm ; Germ Layers ; Pluripotent Stem Cells ; Primates
    Language English
    Publishing date 2022-04-18
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2375354-7
    ISSN 1875-9777 ; 1934-5909
    ISSN (online) 1875-9777
    ISSN 1934-5909
    DOI 10.1016/j.stem.2022.03.014
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  8. Article ; Online: Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

    Kara, Neesha / Krueger, Felix / Rugg-Gunn, Peter / Houseley, Jonathan

    PLoS biology

    2021  Volume 19, Issue 3, Page(s) e3000886

    Abstract: Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which ... ...

    Abstract Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.
    MeSH term(s) 3' Untranslated Regions/genetics ; Animals ; DNA/chemistry ; DNA/genetics ; DNA Breaks, Double-Stranded ; DNA Repair/genetics ; DNA Replication/genetics ; DNA Replication/physiology ; DNA-Binding Proteins/metabolism ; Genome/genetics ; Humans ; Sequence Analysis, DNA/methods
    Chemical Substances 3' Untranslated Regions ; DNA-Binding Proteins ; DNA (9007-49-2)
    Language English
    Publishing date 2021-03-24
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2126776-5
    ISSN 1545-7885 ; 1544-9173
    ISSN (online) 1545-7885
    ISSN 1544-9173
    DOI 10.1371/journal.pbio.3000886
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  9. Article ; Online: The application of cell surface markers to demarcate distinct human pluripotent states.

    Goodwin, Jacob / Laslett, Andrew L / Rugg-Gunn, Peter J

    Experimental cell research

    2019  Volume 387, Issue 1, Page(s) 111749

    Abstract: Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of ... ...

    Abstract Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of the pluripotency spectrum are naïve and primed hPSC, whereby naïve hPSC grown in stringent conditions recapitulate features of the preimplantation human embryo, and the conventionally grown primed hPSC align closer to the early postimplantation embryo. Investigating these cell types will help to define the mechanisms that control early development and should provide new insights into stem cell properties such as cell identity, differentiation and reprogramming. Monitoring cell surface marker expression provides a valuable approach to resolve complex cell populations, to directly compare between cell types, and to isolate viable cells for functional experiments. This review discusses the discovery and applications of cell surface markers to study human pluripotent cell types with a particular focus on the transitions between naïve and primed states. Highlighted areas for future study include the potential functions for the identified cell surface proteins in pluripotency, the production of new high-quality monoclonal antibodies to naïve-specific protein epitopes and the use of cell surface markers to characterise subpopulations within pluripotent states.
    MeSH term(s) Animals ; Biomarkers/metabolism ; Cell Differentiation/physiology ; Embryonic Development/physiology ; Embryonic Stem Cells/metabolism ; Humans ; Membrane Proteins/metabolism ; Pluripotent Stem Cells/metabolism
    Chemical Substances Biomarkers ; Membrane Proteins
    Language English
    Publishing date 2019-11-30
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 1493-x
    ISSN 1090-2422 ; 0014-4827
    ISSN (online) 1090-2422
    ISSN 0014-4827
    DOI 10.1016/j.yexcr.2019.111749
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  10. Article ; Online: Identifying Human Naïve Pluripotent Stem Cells - Evaluating State-Specific Reporter Lines and Cell-Surface Markers.

    Collier, Amanda J / Rugg-Gunn, Peter J

    BioEssays : news and reviews in molecular, cellular and developmental biology

    2018  Volume 40, Issue 5, Page(s) e1700239

    Abstract: Recent reports that human pluripotent stem cells can be captured in a spectrum of states with variable properties has prompted a re-evaluation of how pluripotency is acquired and stabilised. The latest additions to the stem cell hierarchy open up ... ...

    Abstract Recent reports that human pluripotent stem cells can be captured in a spectrum of states with variable properties has prompted a re-evaluation of how pluripotency is acquired and stabilised. The latest additions to the stem cell hierarchy open up opportunities for understanding human development, reprogramming, and cell state transitions more generally. Many of the new cell lines have been collectively termed 'naïve' human pluripotent stem cells to distinguish them from the conventional 'primed' cells. Here, several transcriptional and epigenetic hallmarks of human pluripotent states in the recently described cell lines are reviewed and evaluated. Methods to derive and identify human naïve pluripotent stem cells are also discussed, with a focus on the uses and future developments of state-specific reporter cell lines and cell-surface proteins. Finally, opportunities and uncertainties in naïve stem cell biology are highlighted, and the current limitations of human naïve pluripotent stem cells considered, particularly in the context of differentiation.
    MeSH term(s) Cell Differentiation/genetics ; Cell Differentiation/physiology ; Cell Line ; Cellular Reprogramming/genetics ; Cellular Reprogramming/physiology ; Embryonic Stem Cells/cytology ; Embryonic Stem Cells/metabolism ; Epigenomics ; Humans ; Pluripotent Stem Cells/metabolism
    Language English
    Publishing date 2018-03-25
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 50140-2
    ISSN 1521-1878 ; 0265-9247
    ISSN (online) 1521-1878
    ISSN 0265-9247
    DOI 10.1002/bies.201700239
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