Publications by authors named "Kathy K Niakan"

31 Publications

TRF2-independent chromosome end protection during pluripotency.

Nature 2021 01 25;589(7840):103-109. Epub 2020 Nov 25.

The Francis Crick Institute, London, UK.

Mammalian telomeres protect chromosome ends from aberrant DNA repair. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.
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http://dx.doi.org/10.1038/s41586-020-2960-yDOI Listing
January 2021

Human Embryogenesis: A Comparative Perspective.

Annu Rev Cell Dev Biol 2020 10;36:411-440

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom; email:

Understanding human embryology has historically relied on comparative approaches using mammalian model organisms. With the advent of low-input methods to investigate genetic and epigenetic mechanisms and efficient techniques to assess gene function, we can now study the human embryo directly. These advances have transformed the investigation of early embryogenesis in nonrodent species, thereby providing a broader understanding of conserved and divergent mechanisms. Here, we present an overview of the major events in human preimplantation development and place them in the context of mammalian evolution by comparing these events in other eutherian and metatherian species. We describe the advances of studies on postimplantation development and discuss stem cell models that mimic postimplantation embryos. A comparative perspective highlights the importance of analyzing different organisms with molecular characterization and functional studies to reveal the principles of early development. This growing field has a fundamental impact in regenerative medicine and raises important ethical considerations.
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http://dx.doi.org/10.1146/annurev-cellbio-022020-024900DOI Listing
October 2020

Initiation of a conserved trophectoderm program in human, cow and mouse embryos.

Nature 2020 11 23;587(7834):443-447. Epub 2020 Sep 23.

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London, UK.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human and cow. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.
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http://dx.doi.org/10.1038/s41586-020-2759-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116563PMC
November 2020

The BCL-2 pathway preserves mammalian genome integrity by eliminating recombination-defective oocytes.

Nat Commun 2020 05 25;11(1):2598. Epub 2020 May 25.

Sex Chromosome Biology Laboratory, The Francis Crick Institute, London, NW1 1AT, UK.

DNA double-strand breaks (DSBs) are toxic to mammalian cells. However, during meiosis, more than 200 DSBs are generated deliberately, to ensure reciprocal recombination and orderly segregation of homologous chromosomes. If left unrepaired, meiotic DSBs can cause aneuploidy in gametes and compromise viability in offspring. Oocytes in which DSBs persist are therefore eliminated by the DNA-damage checkpoint. Here we show that the DNA-damage checkpoint eliminates oocytes via the pro-apoptotic BCL-2 pathway members Puma, Noxa and Bax. Deletion of these factors prevents oocyte elimination in recombination-repair mutants, even when the abundance of unresolved DSBs is high. Remarkably, surviving oocytes can extrude a polar body and be fertilised, despite chaotic chromosome segregation at the first meiotic division. Our findings raise the possibility that allelic variants of the BCL-2 pathway could influence the risk of embryonic aneuploidy.
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http://dx.doi.org/10.1038/s41467-020-16441-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248069PMC
May 2020

IGF1-mediated human embryonic stem cell self-renewal recapitulates the embryonic niche.

Nat Commun 2020 02 7;11(1):764. Epub 2020 Feb 7.

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK.

Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.
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http://dx.doi.org/10.1038/s41467-020-14629-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005693PMC
February 2020

Author Correction: Human germline genome editing.

Nat Cell Biol 2020 Jan;22(1):135

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41556-019-0451-xDOI Listing
January 2020

Human germline genome editing.

Nat Cell Biol 2019 12 4;21(12):1479-1489. Epub 2019 Dec 4.

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London, UK.

With the advent of efficient, easy-to-use genome editing by CRISPR-Cas9, editing human embryos is now possible, providing tremendous opportunities to study gene function and cell fate in early human development. The technique can also be used to modify the human germline. Unresolved questions about pre-implantation human development could be addressed by basic research using CRISPR-Cas9. In this Perspective, we discuss advances in human genome editing and consider ethical questions and potential clinical implications of this technology.
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http://dx.doi.org/10.1038/s41556-019-0424-0DOI Listing
December 2019

Effects of thyroid hormone on mitochondria and metabolism of human preimplantation embryos.

Stem Cells 2020 03 26;38(3):369-381. Epub 2019 Dec 26.

Division of Women's and Children's Health, Faculty of Life Sciences and Medicine, King's College London and Assisted Conception Unit, Guy's Hospital, London, UK.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10  M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0 platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.
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http://dx.doi.org/10.1002/stem.3129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064942PMC
March 2020

SETDB1 Links the Meiotic DNA Damage Response to Sex Chromosome Silencing in Mice.

Dev Cell 2018 12 1;47(5):645-659.e6. Epub 2018 Nov 1.

Sex Chromosome Biology Laboratory, The Francis Crick Institute, London NW1 1AT, UK. Electronic address:

Meiotic synapsis and recombination ensure correct homologous segregation and genetic diversity. Asynapsed homologs are transcriptionally inactivated by meiotic silencing, which serves a surveillance function and in males drives meiotic sex chromosome inactivation. Silencing depends on the DNA damage response (DDR) network, but how DDR proteins engage repressive chromatin marks is unknown. We identify the histone H3-lysine-9 methyltransferase SETDB1 as the bridge linking the DDR to silencing in male mice. At the onset of silencing, X chromosome H3K9 trimethylation (H3K9me3) enrichment is downstream of DDR factors. Without Setdb1, the X chromosome accrues DDR proteins but not H3K9me3. Consequently, sex chromosome remodeling and silencing fail, causing germ cell apoptosis. Our data implicate TRIM28 in linking the DDR to SETDB1 and uncover additional factors with putative meiotic XY-silencing functions. Furthermore, we show that SETDB1 imposes timely expression of meiotic and post-meiotic genes. Setdb1 thus unites the DDR network, asynapsis, and meiotic chromosome silencing.
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http://dx.doi.org/10.1016/j.devcel.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286383PMC
December 2018

Human Pre-gastrulation Development.

Curr Top Dev Biol 2018 13;128:295-338. Epub 2018 Feb 13.

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London, United Kingdom. Electronic address:

Understanding the progression of early human embryonic development prior to implantation is of fundamental biological importance. Greater insights into early developmental events may lead to clinical improvements, not only via the establishment of novel stem cell models with increased potential or more physiological relevance, but also by uncovering some underlying causes of infertility, miscarriages, and developmental disorders. The majority of human embryos available for study are those donated to research once they are surplus to family building following in vitro fertilization, though in some countries it is also possible to create embryos using donated gametes. As human embryo development is surprisingly inefficient, with only 40% reaching the blastocyst stage in vitro (French, Sabanegh, Goldfarb, & Desai, 2010; Gardner, Lane, Stevens, Schlenker, & Schoolcraft, 2000), many embryos may not develop to a stage suitable for study. Where legally permitted, the oversight of human embryo research is subject to either ethics approval from a local institutional review board (i.e., China and the United States) or both a national regulator as well as a regional research ethics committee (i.e., the United Kingdom). The study of human development has historically been by necessity comparative, relying on model organisms and stem cell lines to inform analyses. Preimplantation mouse and human embryos in particular exhibit remarkably similar gross morphologies at these early stages of development, although key differences have been identified in gene expression patterns and developmental timing. While recent advances in high-resolution transcriptomic analyses at the single cell level have improved our capability to interrogate expression patterns directly in the human embryo, we still lack an understanding of basic molecular events in the human embryo, including how the first cell lineages become specified. Here, we present a current overview of the major developmental events during human preimplantation development, from fertilization to delineation of the embryonic and extraembryonic lineages prior to implantation. Comparisons to both the mouse and alternative models are included where these have formed the basis for similar investigations in a human context.
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http://dx.doi.org/10.1016/bs.ctdb.2017.11.004DOI Listing
May 2019

Genome editing reveals a role for OCT4 in human embryogenesis.

Nature 2017 10 20;550(7674):67-73. Epub 2017 Sep 20.

Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
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http://dx.doi.org/10.1038/nature24033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815497PMC
October 2017

Jmjd2c facilitates the assembly of essential enhancer-protein complexes at the onset of embryonic stem cell differentiation.

Development 2017 02 13;144(4):567-579. Epub 2017 Jan 13.

Institute of Reproductive and Developmental Biology, Faculty of Medicine, Imperial College London, London W12 0NN, UK

Jmjd2 H3K9 demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity. However, little is known about their importance at the exit of ESC pluripotency. Here, we reveal that Jmjd2c facilitates this process by stabilising the assembly of mediator-cohesin complexes at lineage-specific enhancers. Functionally, we show that Jmjd2c is required in ESCs to initiate appropriate gene expression programs upon somatic multi-lineage differentiation. In the absence of Jmjd2c, differentiation is stalled at an early post-implantation epiblast-like stage, while -knockout ESCs remain capable of forming extra-embryonic endoderm derivatives. Dissection of the underlying molecular basis revealed that Jmjd2c is re-distributed to lineage-specific enhancers during ESC priming for differentiation. Interestingly, Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a (also known as Ehmt2), independently of its H3K9-modifying activity. Loss of Jmjd2c abrogates G9a recruitment and further destabilises loading of the mediator and cohesin components Med1 and Smc1a at newly activated and poised enhancers in ESC-derived epiblast-like cells. These findings unveil Jmjd2c and G9a as novel enhancer-associated factors, and implicate Jmjd2c as a molecular scaffold for the assembly of essential enhancer-protein complexes with an impact on timely gene activation.
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http://dx.doi.org/10.1242/dev.142489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312034PMC
February 2017

Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.

Nature 2016 06 8;534(7607):383-6. Epub 2016 Jun 8.

Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Biomedicine West Wing, Centre for Life, Times Square, Newcastle upon Tyne NE1 3BZ, UK.

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.
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http://dx.doi.org/10.1038/nature18303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131843PMC
June 2016

Self-organization of the human embryo in the absence of maternal tissues.

Nat Cell Biol 2016 06 4;18(6):700-708. Epub 2016 May 4.

Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience; Downing Street, Cambridge, CB2 3DY, UK.

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5049689PMC
http://dx.doi.org/10.1038/ncb3347DOI Listing
June 2016

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

Development 2015 Oct;142(20):3613

There were errors published in Development 142, 3151-3165.In the issue published online on 22 September 2015, Fig. 3 was mislabelled: panels A, B, C and D should have been B, C, D and A, respectively. In the legend, the text prior to ‘(A) Cytoscape enrichment map…’ should not have been included. The correct version of the figure and legend now appear online and in print.We apologise to the authors and readers for this mistake.
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http://dx.doi.org/10.1242/dev.131235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631772PMC
October 2015

Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.

Development 2015 Sep 20;142(18):3151-65. Epub 2015 Aug 20.

Human Embryology and Stem Cell Laboratory, The Francis Crick Institute, Mill Hill Laboratory, London NW7 1AA, UK

Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.
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http://dx.doi.org/10.1242/dev.123547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582176PMC
September 2015

Gata6 potently initiates reprograming of pluripotent and differentiated cells to extraembryonic endoderm stem cells.

Genes Dev 2015 Jun;29(12):1239-55

Mill Hill Laboratory, The Francis Crick Institute, London NW7 1AA, United Kingdom;

Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.
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http://dx.doi.org/10.1101/gad.257071.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4495396PMC
June 2015

Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.

Stem Cells 2015 Sep 23;33(9):2712-25. Epub 2015 Jun 23.

Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.

During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
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http://dx.doi.org/10.1002/stem.2067DOI Listing
September 2015

Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells.

Nat Protoc 2013 Jun 2;8(6):1028-41. Epub 2013 May 2.

The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge, UK.

At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2-3 weeks.
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http://dx.doi.org/10.1038/nprot.2013.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927835PMC
June 2013

Analysis of human embryos from zygote to blastocyst reveals distinct gene expression patterns relative to the mouse.

Dev Biol 2013 Mar 19;375(1):54-64. Epub 2012 Dec 19.

The Howard Hughes Medical Institute, Harvard Stem Cell Institute and the Department of Stem Cell and Regenerative Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138, USA.

Early mammalian embryogenesis is controlled by mechanisms governing the balance between pluripotency and differentiation. The expression of early lineage-specific genes can vary significantly between species, with implications for developmental control and stem cell derivation. However, the mechanisms involved in patterning the human embryo are still unclear. We analyzed the appearance and localization of lineage-specific transcription factors in staged preimplantation human embryos from the zygote until the blastocyst. We observed that the pluripotency-associated transcription factor OCT4 was initially expressed in 8-cell embryos at 3 days post-fertilization (dpf), and restricted to the inner cell mass (ICM) in 128-256 cell blastocysts (6dpf), approximately 2 days later than the mouse. The trophectoderm (TE)-associated transcription factor CDX2 was upregulated in 5dpf blastocysts and initially coincident with OCT4, indicating a lag in CDX2 initiation in the TE lineage, relative to the mouse. Once established, the TE expressed intracellular and cell-surface proteins cytokeratin-7 (CK7) and fibroblast growth factor receptor-1 (FGFR1), which are thought to be specific to post-implantation human trophoblast progenitor cells. The primitive endoderm (PE)-associated transcription factor SOX17 was initially heterogeneously expressed in the ICM where it co-localized with a sub-set of OCT4 expressing cells at 4-5dpf. SOX17 was progressively restricted to the PE adjacent to the blastocoel cavity together with the transcription factor GATA6 by 6dpf. We observed low levels of Laminin expression in the human PE, though this basement membrane component is thought to play an important role in mouse PE cell sorting, suggesting divergence in differentiation mechanisms between species. Additionally, while stem cell lines representing the three distinct cell types that comprise a mouse blastocyst have been established, the identity of cell types that emerge during early human embryonic stem cell derivation is unclear. We observed that derivation from plating intact human blastocysts resulted predominantly in the outgrowth of TE-like cells, which impairs human embryonic stem cell derivation. Altogether, our findings provide important insight into developmental patterning of preimplantation human embryos with potential consequences for stem cell derivation.
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http://dx.doi.org/10.1016/j.ydbio.2012.12.008DOI Listing
March 2013

Pairing of homologous regions in the mouse genome is associated with transcription but not imprinting status.

PLoS One 2012 3;7(7):e38983. Epub 2012 Jul 3.

Epigenetics Programme, The Babraham Institute, Cambridge, United Kingdom.

Although somatic homologous pairing is common in Drosophila it is not generally observed in mammalian cells. However, a number of regions have recently been shown to come into close proximity with their homologous allele, and it has been proposed that pairing might be involved in the establishment or maintenance of monoallelic expression. Here, we investigate the pairing properties of various imprinted and non-imprinted regions in mouse tissues and ES cells. We find by allele-specific 4C-Seq and DNA FISH that the Kcnq1 imprinted region displays frequent pairing but that this is not dependent on monoallelic expression. We demonstrate that pairing involves larger chromosomal regions and that the two chromosome territories come close together. Frequent pairing is not associated with imprinted status or DNA repair, but is influenced by chromosomal location and transcription. We propose that homologous pairing is not exclusive to specialised regions or specific functional events, and speculate that it provides the cell with the opportunity of trans-allelic effects on gene regulation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038983PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3389011PMC
December 2012

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states.

Development 2012 Aug 12;139(16):2866-77. Epub 2012 Jul 12.

The Anne McLaren Laboratory for Regenerative Medicine, Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, UK.

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.
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http://dx.doi.org/10.1242/dev.078519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3403099PMC
August 2012

Human pre-implantation embryo development.

Development 2012 Mar;139(5):829-41

Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK.

Understanding human pre-implantation development has important implications for assisted reproductive technology (ART) and for human embryonic stem cell (hESC)-based therapies. Owing to limited resources, the cellular and molecular mechanisms governing this early stage of human development are poorly understood. Nonetheless, recent advances in non-invasive imaging techniques and molecular and genomic technologies have helped to increase our understanding of this fascinating stage of human development. Here, we summarize what is currently known about human pre-implantation embryo development and highlight how further studies of human pre-implantation embryos can be used to improve ART and to fully harness the potential of hESCs for therapeutic goals.
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http://dx.doi.org/10.1242/dev.060426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274351PMC
March 2012

BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.

Cell Stem Cell 2011 Aug;9(2):144-55

The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge CB2 0SZ, UK.

BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.
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http://dx.doi.org/10.1016/j.stem.2011.06.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567433PMC
August 2011

Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal.

Genes Dev 2010 Feb;24(3):312-26

Stowers Medical Institute, Harvard University, Cambridge, Massachusetts 02138, USA.

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.
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http://dx.doi.org/10.1101/gad.1833510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2811832PMC
February 2010

Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.

Science 2008 Aug 31;321(5893):1218-21. Epub 2008 Jul 31.

Harvard Stem Cell Institute, Stowers Medical Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patient's disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.
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http://dx.doi.org/10.1126/science.1158799DOI Listing
August 2008

Novel role for the orphan nuclear receptor Dax1 in embryogenesis, different from steroidogenesis.

Mol Genet Metab 2006 Jul 8;88(3):261-71. Epub 2006 Feb 8.

Department of Human Genetics, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

Cytomegalic adrenal hypoplasia congenita (AHC) is an X-linked disease caused by mutations in DAX1-encoding gene NR0B1, previously thought to function primarily in steroidogenesis. We sought to determine the expression pattern for Dax1 along with known network partners in early embryogenesis and to determine a steroidogenic capacity for the embryo prior to the establishment of the urogenital ridge at embryonic day 9 (E9). Here, we report that murine Dax1 is a unique marker in early embryonic development, distinguishing the extraembryonic (proximal) endoderm from the remainder of the developing embryo. We showed that Wilms tumor 1, steroidogenic factor 1, and estrogen receptor beta were expressed throughout the embryo, but the progesterone, estrogen alpha and androgen receptors, cytochrome P450 (Cyp11a1) and Nur77 were not observed in any of the embryonic layers. Lack of Cyp11A1 expression at this stage confirmed an absence of inherent steroidogenic capacity for the early embryo. The role of Nr0b1 in embryonic stem (ES) cells was investigated using siRNA knockdown, resulting in differentiation toward endoderm-like fate. Nr0b1 conditional knockout in ES cells led to differentiation, confirming our knockdown results. Our investigations suggest that Nr0b1 functions in a novel role in the maintenance of a relatively undifferentiated state. Our results further suggest that the failure of conventional murine Nr0b1 knockout attempts may be due to disregulated differentiation.
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http://dx.doi.org/10.1016/j.ymgme.2005.12.010DOI Listing
July 2006

IL1RAPL1 is associated with mental retardation in patients with complex glycerol kinase deficiency who have deletions extending telomeric of DAX1.

Hum Mutat 2004 Sep;24(3):273

Department of Pediatrics, UCLA School of Medicine, Los Angeles, California 90095-7088, USA.

IL1RAPL1 (interleukin-1 receptor accessory protein-like, gene 1) has recently been shown to be mutated in patients with X-linked mental retardation. Clinical experience has suggested that patients with the contiguous gene syndrome, complex glycerol kinase deficiency (cGKD), will have mental retardation (MR) if they have deletions extending from the GK gene into the DMD gene and/or involving a significant extension telomeric from DAX1. We examined cell lines from patients with cGKD whose clinical features would be informative and would allow us to determine if IL1RAPL1 deletions can help to explain the MR in patients with deletions extending telomeric from DAX1. Our results showed that nearly all patients with deletions involving DAX1, but not DMD, had MR if IL1RAPL1 was deleted. If ILIRAPLI and DMD were intact, the patients with DAX1 deletions only rarely had normal development. Deletions in DNA from patients with cGKD who exhibited MR and had normal IL1RAPL1 all involved the GK and DMD genes. Our data are consistent with the association of IL1RAPL1 gene deletion and MR in the majority of patients with cGKD and deletions extending telomeric from DAX1.
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http://dx.doi.org/10.1002/humu.9269DOI Listing
September 2004