Mechanisms of lineage specification in human embryos and stem cells

Lead Research Organisation: MRC National Inst for Medical Research

Abstract

To understand how a less specialized cell becomes more specialized, we are using embryonic stem (ES) cells that are taken from the early human or mouse embryo and grown in a dish. ES cells are able to renew themselves and can give rise to other cell types. ES cells can be used to uncover fundamental aspects of early cell fate choices, for example how a cell chooses to remain embryonic or to differentiate. We have established ES cells that can be manipulated in a controlled way to determine the sequence of events at the gene and protein level as a stem cell undergoes directed differentiation. We predict that certain genes act dominantly to directly turn on and off genes and that this unique dual function influences cellular differentiation. We will change the environment and type of starting stem cell to determine if this has an influence on cellular differentiation. We predict that stem cells derived from embryos that are developmentally more mature will respond distinctly to differentiation signals compared to ES cells. By defining the relevant genes expressed in early human embryos, we intend to establish alternative clinically useful models of human stem cells and to test unique and conserved mechanisms of mammalian development.

Technical Summary

The allocation of cells to a specific lineage is regulated by the activities of key signalling pathways and developmentally regulated transcription factors. The focus of our research is to understand the influence of signalling and transcription factors on differentiation during early human development. During preimplantation development totipotent human zygotes differentiate into pluripotent embryonic cells, which form the foetus, and extra-embryonic cells, which form the placenta and yolk sac.
The central question we are addressing is what are the molecular mechanisms that regulate embryonic stem cell pluripotency and how is it disengaged during cellular differentiation? We seek to define the genetic hierarchy acting during differentiation, the influence of extracellular signalling and the extent to which these mechanisms are conserved between humans and mice.
Towards this aim, we are characterizing the gene expression patterns throughout human preimplantation development. We will use this information to modulate conditions for in vitro establishment of alternative stem cells from human embryos, with the aim of expanding the repertoire of clinically relevant cells.
Although we have discovered significant differences between human and mouse preimplantation development, many of the key mechanisms are conserved, including the expression of pluripotency transcription factors Nanog and Oct4. Therefore, to complement our human research and to mechanistically understand how stem cells differentiate, we are using mouse embryonic stem cells (mESCs), which are highly amenable to genetic manipulation. For example, we have generated mESCs that overexpress transcription factors in a controlled (doxycycline) manner, allowing for inducible differentiation. We hypothesize that the unique ability of specific transcription factors to induce ESC differentiation results from a dual function in activation of endoderm genes and simultaneous direct repression of pluripotency genes.
We have developed approaches using growth factors that allow for the generation of self-renewing extra-embryonic endoderm cells without the requirement for gene manipulation. A notable advantage of this approach is potential application to mutant mESCs, allowing genetic study of extra-embryonic endoderm development. Using this approach we seek to further dissect the genetic network and signalling environments that influence stem cell differentiation and to determine whether extra-embryonic stem cells can be derived from human embryonic stem cells. These experiments will provide novel insight into the impact of signal transduction on the pluripotency gene regulatory network.
The molecular basis of these early cell lineage decisions are of fundamental biological importance and have significant clinical implications for infertility, miscarriages, developmental disorders and therapeutic application of stem cells.
 
Description Licence from the HFEA to use CRISPR/Cas9 for human genome editing in basic research has led to several citations in public ethics documents and discussions with policy makers in several countries
Geographic Reach National 
Policy Influence Type Citation in other policy documents
URL http://guide.hfea.gov.uk/guide/ShowPDF.aspx?ID=5966
 
Description Nuffield Council on Bioethics Genome Editing: An Ethical Review
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
 
Description Provided evidence on embryos generated for mitochondrial replacement therapy to the Department of Health
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
URL http://www.hfea.gov.uk/6896.html
 
Description Research Grant
Amount $358,707 (USD)
Funding ID 1-FY11-436 
Organisation March of Dimes Foundation 
Sector Charity/Non Profit
Country United States
Start 06/2011 
End 05/2014
 
Title Newcastle-Crick Human Embryonic Stem Cells 
Description We generated several human embryonic stem cells lines from embryos that had undergone pronuclear transfer as part of a collaboration with the University of Newcastle. 
Type Of Material Cell line 
Provided To Others? No  
Impact The cells lines have been used to investigate mitochondrial carryover following pronuclear transfer to evaluate the therapeutic potential of these methods to prevent the inheritance of mitochondrial disease. We are currently working with the University of Newcastle to deposit these cell lines into the UK Stem Cell Bank. 
 
Title ChIP-sequencing analysis of Gata6 binding in extra embryonic endoderm cells 
Description (1) Microarray analysis of Gata6 overexpressing cells from 12 to 144 hours of doxycycline treatment in mouse embryonic stem (mES) cells compared to uninduced mES cells, embryo-derived XEN cells and Sox7 overexpressing mES cells after 144 hours of doxycycline treatment. (2) ChIP-seq analysis of Gata6 binding 36 hours following doxycycline treatment. (3) ChIP-seq analysis of Gata6 binding in embryo-derived XEN cells. (4) RNA-seq analysis of GATA6 overexpressing cells following 144 hours of induction in hES cells. The data has been deposited into gene expression omnibus: GSE69322 and GSE69323 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, alongside step-wise activation of extraembryonic endoderm genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near both 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 reprogramming factor that can act alone to drive a cell fate switch from diverse cell types. This work led to a publication (PMID: 26109048) and is being used by other research groups. 
URL http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE69322
 
Title Single-cell RNA-seq data from human preimplantation embryos 
Description A database of single-cell RNA-sequencing of human preimplantation embryos was uploaded onto Gene Expression Omnibus (GSE66507). An output database of processed data was also uploaded publicly at Figshare: https://figshare.com/articles/Defining_the_three_cell_lineages_of_the_human_blastocyst_by_single_cell_RNA_seq/1521657 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact This dataset was used to provide fundamental insights into early human development. We elucidate conserved transcriptional programs along with those that are human-specific. Comparisons 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 to mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells. We published these results (PMID: 26293300). The database has been used by several groups, including a recent publication (PMID: 26947977). 
URL https://figshare.com/articles/Defining_the_three_cell_lineages_of_the_human_blastocyst_by_single_cel...
 
Title Single-cell RNA-sequencing analysis of embryos generated by pronuclear transfer 
Description We evaluated the transcriptome of embryos generated by pronuclear transfer to determine how the gene expression of these embryos compares to embryos generated by more conventional IVF approaches. We deposited this database in the publicly accessible Gene Expression Omnibus: GSE76284. This analysis allowed us to determine to what extent this novel technology would affect gene expression. Embryos that were deemed to be chromosomally normal and not aneuploid were indistinguishable from the reference control samples suggesting that the embryos generated by this method are indistinguishable from control samples. Pronuclear transfer has been proposed as a method that could be used to uncouple nuclear DNA inheritance from the inheritance of mitochondrial DNA for mitochondrial replacement therapy. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact We published these results (PMID: 27281217). 
 
Description Assisted Conception Unit, Guy's and St Thomas' Hospital 
Organisation Guy's and St Thomas' NHS Foundation Trust
Department Assisted Conception Unit (ACU)
Country United Kingdom 
Sector Hospitals 
PI Contribution We are providing molecular biology and computational biology expertise as part of this collaboration.
Collaborator Contribution The Assisted Conception Unit at Guy's and St Thomas' Hospital are providing us with embryo and expertise in embryo manipulation techniques.
Impact We are currently completing a manuscript for publication.
Start Year 2013
 
Description Bourn Hall Clinic 
Organisation Bourn Hall Clinic
Country United Kingdom 
Sector Hospitals 
PI Contribution We have contributed to this collaboration by providing molecular techniques (single-cell RNA-seq, immunohistochemistry) and analysis that has resulted in novel discoveries transforming our understanding of early human biology. We are also contributing to this collaboration by developing methods for the establishment of novel human stem cells (both embryonic and extra embryonic) that could in the future be used in therapeutic application to improve human health.
Collaborator Contribution Bourn Hall Clinic provides us with material (human preimplantation embryos) and expertise in embryo culture techniques.
Impact This collaboration has led to the publication of a paper (Blakeley et al., Development 2015). We discovered that there are many genes that are uniquely expressed in the human, but not mouse, preimplantation embryo and that are also absent in existing human embryonic stem cells. In order to understand the function of these genes that are uniquely expressed in the human embryo, we have been granted a research licence by the Human Fertilisation and Embryology Authority to perform genome editing on human embryos using novel methods of CRISPR-Cas9. We are currently preparing two additional publications as part of this collaboration.
Start Year 2009
 
Description Newcastle Centre for Life 
Organisation Newcastle University
Country United Kingdom 
Sector Academic/University 
PI Contribution The Newcastle Centre for Life have a long standing interest in developing novel in vitro fertilisation therapies for currently incurable mitochondrial disease. My research team has made significant contributions to a collaborative project with the Newcastle Centre for Life including performing single-cell RNA-sequencing and training nurses, postdocs and PhD students in micromanipulation. We have used these novel cutting edge methods of single-cell RNA-sequencing to determine how the gene expression pattern of embryos generated by pronuclear transfer compare to embryos generated by more conventional in vitro fertilisation approaches. To evaluate the therapeutic potential of the embryos generated by pronuclear transfer we have generated several Newcastle-Crick human embryonic stem cell lines that are currently being deposited into the UK Stem Cell Bank.
Collaborator Contribution The Newcastle Centre for Life transported embryos generated by pronuclear transfer embryos to our lab. Our collaborators optimised the pronuclear transfer techniques and performed mitochondrial carryover analysis of the embryos dissected in our lab.
Impact This is a multi-disciplinary collaboration involving clinicians and embryologists at the Newcastle Centre for Life and our expertise in single-cell genomics and stem cells. We have evaluated the impact on embryogenesis of a potential therapy to cure childhood mitochondrial diseases. We developed novel methods to analyse transcriptomics datasets and submitted our data to the HFEA and UK Department of Health ahead of a positive vote by Parliament on February 3rd 2015 to change laws related to new IVF technology to prevent transmission of serious mitochondrial disease. We are co-authors on a manuscript that is under review at Nature describing these findings.
Start Year 2014
 
Description The Bridge Centre 
Organisation London Bridge Fertility, Gynaecology and Genetics Centre
Country United Kingdom 
Sector Hospitals 
PI Contribution We will be providing expertise in genome editing and molecular characterisation of embryos donated by couples who have undergone infertility treatment and no longer require their surplus embryos. We will also be providing expertise in the derivation of human stem cells.
Collaborator Contribution The Bridge Centre will be providing us with material (human preimplantation embryos) donated to our research project. Moreover, the Centre's expertise in karyotyping of human embryos will be utilised to further characterise the samples used in the research program.
Impact None yet - only recently have embryos been consented for this project.
Start Year 2016
 
Description Dialogue with international policy makers 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact I have engaged with international policy makers including the Norwegian Directorate of Health, Singapore Ministry of Health, German Department for Economic Affairs, Energy and Global Issues and the French Science and Technology Department to discuss our work and the regulatory oversight including the application for the HFEA research licence and ethics approval.
Year(s) Of Engagement Activity 2016,2017
 
Description Lecturer at King's College to undergraduates and MSc students 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Lecturer on early human development and stem cells for a course on Introduction to Stem Cells at King's College London. This sparked several questions afterwards and students had approached me to apply to the Francis Crick Institute PhD programme.
Year(s) Of Engagement Activity 2015
 
Description Medical Research Council, Human Tissue Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact A discussion with the Human Tissue Forum including members of the MRC's Regulatory Support Centre about our experience with the application to the HFEA and Health Research Authority Research Ethics Committee.
Year(s) Of Engagement Activity 2016
 
Description Press release - related to the development of a novel therapy to prevent the transmission of inherited mitochondrial diseases 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The publication of our collaborative work to develop a novel therapy to prevent the transmission of inherited mitochondrial diseases has been of widespread public interest.
Year(s) Of Engagement Activity 2016
 
Description Project Educational Trust 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A diverse audience attended the Conference which introduced how genome editing using CRISPR/Cas9 could be used to understand fundamental principles of human development. This was followed by a public discussion about the use of the technology and several members of the audience continued the discussion afterwards saying that it had increased their understanding of this research area.
Year(s) Of Engagement Activity 2016
URL http://www.progress.org.uk/conference2016
 
Description Science Media Centre Briefing on Human Genome Editing 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Our recent Human Fertilisation and Embryology (HFEA) licence (R0162) to genetically modify human embryos for research attracted widespread media and policy interest. I have worked with the Science Media Centre to discuss our research objectives in relation to human embryo genome editing. I have worked with leading journals and news media to inform the public about the rational behind our research and potential benefits. We generated a web link to allow the public to view all of the HFEA and Research Ethics Committee approved patient information sheets and consent documents and background information related to the licence. The work was widely reported in the UK (e.g. Telegraph, Independent, Guardian, BBC), US (e.g. NY Times, LA Times, Washington Post) and international press of many countries.
Year(s) Of Engagement Activity 2016
 
Description Science Media Centre organised press release - the development of methods to investigate early human postimplantation development in vitro 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Our collaborative work that led to the development of methods to investigate early human post implantation development in vitro led to widespread media interest. It also led to several debates (Project Educational Trust, Nuffield Council on Bioethics) that increase the awareness of the impact of this work.
Year(s) Of Engagement Activity 2016
 
Description UK Department of Health Seminar Series 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact A presentation of our research goals and preliminary data sparked questions about our research and increased transparency about our approaches. We have recently been asked to provide evidence to the House of Commons' Science and Technology Select Committee: Genomics and Genome Editing inquiry.
Year(s) Of Engagement Activity 2016
 
Description University of Oxford MSc Course on Human Embryology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Lectured on Human Development and Stem Cells for an MSc course on Human Embryology at the University of Oxford. The lecture sparked a discussion afterwards on the use of human genome editing.
Year(s) Of Engagement Activity 2015
 
Description University of the Third Age Public Engagement 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact My presentation lead to several questions related to the research and to public policy.

Several members of the audience approached me afterwards to ask for more information and for the slides from the talk.
Year(s) Of Engagement Activity 2014
 
Description Young Embryologist Network Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact PhD students organised the conference where I discussed our research. This led to discussions afterwards and increase interest in the subject area with a number of students wanting to pursue a postdoc in the field.
Year(s) Of Engagement Activity 2016