Comparative transcriptional control of establishment, maintenance and collapse of naive pluripotency in rodents and primates in vivo

Lead Research Organisation: University of Cambridge
Department Name: Wellcome Trust - MRC Cam Stem Cell Inst

Abstract

The value of stem cell lines that can multiply in culture and retain the capacity to become any tissue type in the body is immense, both from the point of view of generating animal models for research, and to produce patient-specific human tissue for drug screening or cell replacement therapy. Stem cells derived from the founder tissue of the foetus in the early mouse embryo, known as pluripotent embryonic stem (ES) cells, have been proven to generate any type of adult tissue, including the germ cells, by observing their contribution to animals produced after integrating ES cells into a host embryo. Human ES cells, however, have characteristics distinct from mouse ES cells; this is evident in their molecular attributes, morphology and culture requirements. In fact, human ES cells resemble much more closely the cells found in more mature mouse embryos that have already implanted in the uterus and are considered to be 'primed' for differentiation. These cells are harder to grow and are more restricted in their differentiation repertoire compared with mouse ES cells. In the interests of therapeutic relevance, it would be highly desirable to understand how to capture cells from human embryos that are more akin to mouse ES cells. Although attempts have been made to produce such cells, either by manipulating the culture conditions in which they are grown, or by means of 'reprogramming' strategies that can revert adult cell types to a more embryonic condition, it has not been possible to capture human cells that can thrive in such a state. We believe that the only way to overcome this obstacle is to understand how these founder embryonic cells are formed in mouse embryos, from which true ES cell lines can be readily obtained. The ultimate goal is to use this knowledge as a blueprint to probe the development of early human embryos (or non-human primate embryos as a model for humans) and exploit alternative pathways to capture similar pluripotent cells from primates. In addition to discovering how to derive the ideal type of stem cells from human embryos, this knowledge will be used to generate useful cells from adult tissues and patient samples. In this project we plan to expand on our previous work to produce a detailed molecular portrait of mouse embryos before, during and after the stage in animal development when pluripotent cells are naturally produced. We will make use of genetic modification strategies to explore the importance of master control genes in this process. In addition, we can add highly specific chemicals to the culture medium that activate or suppress different cellular behaviours to reveal how these control genes are turned on or off. We have already begun to identify molecular differences between rodent (mouse) and primate (marmoset) embryos. We will extend this concept and then test how marmoset embryos respond to external signals that we predict may be beneficial to development. We will use this information to target specific pathways that could be enhanced or inhibited to allow pluripotent cells to be captured, thereby providing the essential starting point to create useful tissues for drug screening and, ultimately, cell replacement therapy.

Technical Summary

To determine the molecular requirements for naïve pluripotency, we will produce a quantitative profile of transcription factor expression patterns and interactions during the establishment of epiblast in the murine embryo using single cell RNA-seq and targeted qRT-PCR. To determine the essential transcriptional network for establishing and maintaining naïve pluripotency, we will probe the phenotypic effects of deletion of key pluripotency regulators in uninterrupted development and during embryonic diapause. The induction of gene expression by extrinsic signals will also be interrogated by application of agonists and antagonists to candidate signalling pathways to cultured embryos. The state of naïve pluripotency during normal development is transient, and the epiblast rapidly becomes primed after implantation incurring a loss of naïve pluripotency. We recently showed that the bHLH transcription factor Tfe3 is exported from the nucleus in epiblast cells soon after implantation, coincident with this state change. We will explore its subcellular localisation by immunohistochemistry during embryonic diapause, when the state of naïve pluripotency is extended. To ascertain whether this interesting intracellular protein shuttling activity is necessary for exit from naïve pluripotency in vivo, we will make use of a novel transgenic mouse line in which Tfe3 protein can be ectopically directed to the nucleus, and characterise the subsequent developmental potential of the epiblast. The functional and molecular blueprint we generate from these combined experiments will be compared with transcriptional profiles and immunohistochemistry of marmoset embryos before and after explant culture, with or without agonists or antagonists of potentially relevant pathways. Ultimately, we aim to attempt to direct the development of primate embryos or isolated cells/tissues to explore the possibility of capturing authentic pluripotent cell lines.

Planned Impact

Who will benefit from this research?
The primary beneficiaries of this project will be scientists engaged in basic developmental biology and stem cell research, especially members of the group and our collaborators. Members of the clinical sector and companies engaged in developing and manufacturing regenerative medicine applications of human ES and iPS cells (e.g. Neusentis, GSK, ACT, Viacyte) will also be interested, as will companies providing tools to support these developments (e.g. LifeTechnologies, TAP Biosystems, Stem Cell Technology), and the Cell Therapy Catapult. Project outputs will also benefit companies developing in vitro assay systems for drug discovery, disease modelling and toxicology (e.g. GSK, AstraZeneca, Cellartis, SC4SM). The ultimate beneficiaries of our work will be clinicians and patients.

How will they benefit from this research?
Preliminary data will be discussed informally in lab meetings and internal seminars and symposia; as the output develops into cohesive results they will be discussed in the wider scientific community. Members of the research group will benefit from the skills developed from this very specialist programme. They will be stretched intellectually because of the nature of the research question and the need to develop novel approaches to processing the material and data. They will be encouraged to present their work informally, at international conferences and in the form of outreach activities. We are confident that this research will be of widespread interest, and therefore publishable in high impact journals. This will assist both post docs to achieve independent positions by the end of the project.
Within the short term, biomedical companies may benefit considerably from the availability of non-human primate models of pluripotent stem cell lines, which we expect to develop within 3 years, that have the potential to be rigorously validated before the protocols we use to develop them have been translated to the human system.
The inevitable expansion of knowledge pertaining to the process and molecular control of early primate development achieved during this project may enable the improvement of culture media for IVF programmes (human and non-human primate) and inform diagnosis and treatment of early developmental defects.
The potential to improve the differentiation capacity of human pluripotent stem cell lines resulting from our molecular characterisation and experimental manipulation of culture regimes will enhance understanding of the signalling requirements for directed exit from pluripotency and is likely to enrich the repertoire of tissues available for drug screening and ultimately, transplantation to patients.

Publications

10 25 50
 
Description Finding out the similarities and differences between the mechanisms of tissue formation and function in various mammalian species has important implications for acquisition of knowledge and development of culture regimes for improved viability of embryos for assisted conception in humans and other mammals. We have performed detailed molecular characterisation of embryonic material and embryonic stem cells to discover the overlap and divergence between species. We add inhibitors or activators of specific signalling pathways to investigate how the proportions of founder tissues can be influenced, since establishment of the placenta and yolk sac in a timely manner is of paramount importance for successful pregnancy. Since embryos are very precious, we also characterise and use embryonic stem cell lines to model certain aspects of early development, such as the beginning of foetal development and specification of tissues required by the adult. We have found that mouse and human embryos differ in their signalling requirements, both for the maintenance of the embryonic stem cell lines, and for the induction of hypoblast, the founder of the yolk sac. Basically, whereas in the mouse embryo FGF signalling is necessary and sufficient for hypoblast formation, in the human embryo Wnt signalling also plays a role.
Exploitation Route The transcriptional profiling datasets are freely available and have been used extensively by other researchers. The web app created by Dr Stirparo is a very accessible tool for visualising the expression of genes in the early embryo. It shows mouse, human and marmoset embryos on the same page with graded colour to show levels of gene expression in each cell type. Our multiple research publications have been highly cited. We also produced a couple of reviews, which have also been used for our own graduate and undergraduate teaching and presenting the questions we address in an easily digestible format during seminars.
Sectors Pharmaceuticals and Medical Biotechnology

URL http://app.stemcells.cam.ac.uk/GRAPPA/
 
Title Comparative gene expression in early mammalian embryos 
Description The tool is an easily accessible website that allows interested parties to view and compare gene expression data for mouse, human and marmoset early embryos. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact Other researchers are able to access this tool to help inform their own studies. It accompanies our recently published re-analysis of the human dataset published by another group in Cell, and we anticipate it will be widely viewed, once our manuscript on comparative transcriptomes of mice, human and marmoset is published. 
URL http://app.stemcells.cam.ac.uk/GRAPPA/
 
Title DNA methylation profiling of human naive embryonic stem cells 
Description Human pluripotent cell lines were derived from blastocyst-stage embryos and propagated in self-renewal conditions that maintain features of naive pluripotency characteristic of mouse embryonic stem cells. Genome-wide DNA methylation status of HNES1 and HNES3 naive and primed cells was assessed with post-bisulfite adapter tagging (PBAT). 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact This collection provides DNA methylation data for the first authentic human embryonic stem cells. The data are available in the public domain under ArrayExpress accession E-MTAB-4462. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4462
 
Title Karyotype analysis of human naive embryonic stem cells 
Description Human pluripotent cell lines were derived from blastocyst-stage embryos and propagated in self-renewal conditions that maintain features of naive pluripotency characteristic of mouse embryonic stem cells. Genomic integrity of the HNES1 cell line was assessed with the Affymetrix CytoScan 750K array. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact This collection provides high-density CGH data for the first authentic human embryonic stem cells. The data are available in the public domain under ArrayExpress accession E-MTAB-4463. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4463
 
Title Pathway atlas of mammalian embryogenesis 
Description We integrated RNA-seq data from mouse and marmoset embryos with 229 annotated KEGG pathways and generated expression maps for pairwise comparison of developmental stages in the two species. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This resource provides a comprehensive collection of pathway maps relating mouse and marmoset embryonic development. 
URL http://pathway-atlas.stemcells.cam.ac.uk
 
Title RNA sequencing of marmoset embryonic development 
Description Transcriptional profiling of common marmoset embryo stages spanning E5.0 to E7.0 was performed by RNA-seq. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This collection provides stage-matched transcriptome data for early lineages of the marmoset embryo, and is the highest quality resource of its kind. The data are available in the public domain under ArrayExpress accession E-MTAB-2959. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2959
 
Title RNA sequencing of mouse embryonic development 
Description Transcriptional profiling of mouse embryos spanning the 8-cell morula stage to E5.5 postimplantation epiblast was performed using lineage-specific RNA-seq. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This collection provides stage-matched transcriptome data for early lineages of the mouse embryo, and is the highest quality resource of its kind. The data are available in the public domain under ArrayExpress accession E-MTAB-2958. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2958
 
Title Reannotation of the common marmoset (Callithrix jacchus) genome 
Description We observed that annotation of marmoset genes was often incorrect at the 3' UTR. Marmoset transcripts were assembled de novo from RNA-seq data, and transcript contigs were mapped to the marmoset reference genome and transcriptome sequences. Newly-assembled transcripts that could be uniquely assigned to a known gene were incorporated into the genome annotation as a new isoform. A total of 10,995 of 20,993 annotated genes were revised in this manner. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This annotation resource is used to update the coordinates of transcribed genes throughout the marmoset genome. The extended annotation may then be used as a reference to quantify gene expression from RNA sequencing experiments. The data are provided in the public domain in the ArrayExpress repository under accession E-MTAB-2959. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2959
 
Title Transcriptional profiling of human naive embryonic stem cells 
Description Human pluripotent cell lines were derived from blastocyst-stage embryos and propagated in self-renewal conditions that maintain features of naive pluripotency characteristic of mouse embryonic stem cells. Transcriptional activity of HNES1, HNES2 and HNES3 cell lines was assessed with RNA-seq. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact This collection provides transcriptome data for the first authentic human embryonic stem cells. The data are available in the public domain under ArrayExpress accession E-MTAB-4461. 
URL https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-4461
 
Description Central Institute for Experimental Animals (CIEA), Kawasaki, Japan 
Organisation Central Institute for Experimental Animals (CIEA)
Department Department of Applied Developmental Biology
Country Japan 
Sector Private 
PI Contribution We undertake detailed molecular analysis of early mammalian embryonic development, and have been able to extend this work to the common marmoset (Callithrix jacchus) through our partnership with the CIEA. The marmoset is an attractive model, as embryos that have developed in utero can be retrieved by non-surgical uterine flush without harming the animal. This provides a high-quality resource for the analysis of early embryogenesis in a non-human primate.
Collaborator Contribution We receive marmoset embryos from our collaborator Dr Erika Sasaki, located at the Central Institute for Experimental Animals (CIEA) in Japan. Dr Sasaki has hosted us in her lab to perform embryo culture experiments and learn related techniques.
Impact Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, Nichols J, Smith A, Bertone P (2015) Lineage-specific profiling delineates the emergence and progression of naïve pluripotency in mammalian embryogenesis. Dev Cell 35: 366-382.
Start Year 2012
 
Description Deutsches Primatenzentrum, Göttingen, Germany 
Organisation Leibniz Association
Department Leibniz Institute for Primate Research
Country Germany 
Sector Academic/University 
PI Contribution We undertake detailed molecular analysis of early mammalian embryonic development, and have been able to extend this work to the common marmoset (Callithrix jacchus) through our partnership with the German Primate Center. The marmoset is an attractive model, as embryos that have developed in utero can be retrieved by non-surgical uterine flush without harming the animal. This provides a high-quality resource for the analysis of early embryogenesis in a non-human primate.
Collaborator Contribution We receive marmoset embryos from our collaborator Rüdiger Behr, located at the German Primate Center in Göttingen, Germany.
Impact Boroviak T, Loos R, Lombard P, Okahara J, Behr R, Sasaki E, Nichols J, Smith A, Bertone P (2015) Lineage-specific profiling delineates the emergence and progression of naïve pluripotency in mammalian embryogenesis. Dev Cell 35: 366-382.
Start Year 2012
 
Description 'Pint of Science' talk, Panton Arms, Cambridge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 'Pint of Science' is a growing international endeavour, in which scientists are invited to present their work in lay terms in a local pub. The subject is then discussed with the audience. Last year was the first time that the organisers also integrated local artists in a new programme called 'Creative Interactions', which provided artistic interpretation of the work of each of the invited speakers.
Year(s) Of Engagement Activity 2015
URL https://pintofscience.co.uk/city/cambridge
 
Description 5th Cambridge Stem Cell International Symposium (organiser) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An international conference featuring diverse areas of stem cell biology and keynote presentations from leaders in the field. The theme of the symposium in 2016 embraces holistic analyses of stem cell biology, incorporating genomics, proteomics and mathematical modeling to highlight approaches beyond traditional single-gene or single-pathway studies.
Year(s) Of Engagement Activity 2016
URL http://www.stemcells.cam.ac.uk/news-events/symposium2016
 
Description Hosting Nuffield Research Placements summer student 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Selected pupils from state schools within the East of England were invited to visit my lab for 4 weeks during the summer holidays. During this time, they conducted a mini project, with supervision and interaction with members of the lab and special facilities.
Year(s) Of Engagement Activity 2015
 
Description Hosting work experience school pupil 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Hosted a secondary school pupil for work experience for one week. During this time, he interacted with members of the lab and institute and observed and participated in some of our current projects.
Year(s) Of Engagement Activity 2015
 
Description Oxbridge Academic Programmes Tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Oxbridge Alumni were invited into the institute and various tours were provided. I demonstrated isolation of early mouse embryos and explained how they are used to isolate embryonic stem cells. The audience came in a series of small groups and asked questions during and after the demonstration.
Year(s) Of Engagement Activity 2015
 
Description Pint of Science Stem Cell Exchanges podcast 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Graduate students recorded podcasts for each scientist participating in 'Pint of Science' and these are all available on the CSCI website.
Year(s) Of Engagement Activity 2017
URL https://www.stemcells.cam.ac.uk/public/podcasts
 
Description Pint of Science stem Cell Exchange 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Local artists and poets were invited to create pieces of work relating to the research performed within the Cambridge Stem Cell Institute. I met with my artist and poet ahead of the main event to explain my work and they each created pieces to reflect the aspects that interested them. The art was showcased in a church/cafe in central Cambridge for a two week period, culminating in an evening event that brought all the participating artists, poets and scientists together to discuss the pieces and perform the poetry.
Year(s) Of Engagement Activity 2017
URL http://www.stemcells.cam.ac.uk/public/past-events-and-projects/2017/exchanges-at-the-stem-cell-insti...
 
Description Public lecture for Cambridge Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was one of the invited public lectures in the annual Cambridge Science Festival in which I presented a lecture outlining the background and state of current knowledge defining the origin, properties and uses of pluripotent stem cells. The audience then asked questions, mostly regarding the medical implications of the work.
Year(s) Of Engagement Activity 2015
URL http://www.sciencefestival.cam.ac.uk/about/past-festivals/2015-cambridge-science-festival
 
Description Stem Cells: Big Data and Personalised Medicine 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact From http://www.sciencefestival.cam.ac.uk/events/stem-cells-big-data-and-personalised-medicine

A panel of biomedical research experts respond to public questions about the complexities of personalised medicine and stem cell research. How are we currently using big data? How can stem cells contribute to diagnostics and drug development? Part of the Cambridge Science Festival, this event invites members of the public to take part in an open discussion about the potential of stem cell research.
Year(s) Of Engagement Activity 2016
URL http://www.sciencefestival.cam.ac.uk/events/stem-cells-big-data-and-personalised-medicine