ELF5-controlled transcriptional networks define stem cell potency and differentiation in the trophoblast compartment

Lead Research Organisation: Babraham Institute
Department Name: Epigenetics

Abstract

Lay summary (4000 chrs) Stem cells are cells that have the potential to reproduce themselves ('self-renew') as well as to form various terminally differentiated cell types of the body. For this reason stem cells are of high interest for biomedical research and regenerative medicine. We are particularly interested in stem cells that contribute exclusively to tissues of the placenta (so-called trophoblast stem cells). These stem cells are of enormous value to study developmental processes at the earliest stages of pregnancy where biological material is not available from humans. In the longer term they may also prove to be valuable for the treatment of pregnancy disorders many of which are based on a defective function of the placenta, i.e. the organ that mediates all nutrition and oxygen supply to the growing baby. A key question is how the expansion and then differentiation of this particular stem cell type is regulated to ensure the formation of a fully functional placenta. The importance of this question is further reinforced by the fact that residual trophoblast stem cells pose a tremendous risk of aggressive tumour formation in the uterus of the mother. We have identified that this mechanism may be self-controlled by a specific molecule that regulates the activity of placental genes, the transcription factor ELF5. This project investigates the molecular basis how ELF5 promotes both, trophoblast stem cell self-renewal as well as differentiation into specific placental cell types. This molecular 'switch' function between promoting the expansion of a specific stem cell population followed by triggering their terminal differentiation is of fundamental importance for a healthy pregnancy (for both mother and baby). It may also prove to be translatable into other cell and organ systems where this molecule is present and that are particularly susceptible to an imbalance between proliferation and differentiation, such as the breast with its susceptibility to develop cancer. Hence understanding the mechanism of ELF5 function may establish a paradigm for molecular pathways that ensure normal reproduction as well as health throughout adult life and ageing.

Technical Summary

Trophoblast stem (TS) cells represent the developmental counterpart to embryonic stem (ES) cells, capable of differentiating into all trophoblast cell types of the placenta. Proper expansion followed by differentiation of the TS cell compartment in vivo is essential for normal development and reproduction. We have recently discovered the transcription factor ELF5 as a master regulator with dual function in the trophoblast lineage. ELF5 is critical for TS cell self-renewal by reinforcing a transcription factor circuit with CDX2 and EOMES. However, sustained ELF5 expression triggers exit from the stem cell niche and commitment to differentiate. This control mechanism may represent an intrinsic limitation to (stem) cell hyperproliferation in the healthy organism. In the current proposal we will investigate how this 'molecular switch' function of ELF5 governs self-renewal and differentiation in the trophoblast compartment. We will determine the transcriptional networks established by the core CDX2/EOMES/ELF5 circuit in TS cells. By integrating these data with the pluripotency networks present in ES cells, as well as with other genetic and epigenetic profiling datasets, we will gain critical insights into the shared and the distinct hallmarks that define the stem cell state in TS versus ES cells, enabling us to correlate genetic and epigenetic profiles with stem cell potency. Further, we will elucidate the mechanistic basis of the differentiation-promoting function of ELF5 by assessing the importance of absolute ELF5 levels, niche environment-dependent ELF5 protein complex components and post-translational modifications that may fine-tune their function. Our data will provide fundamental insights into stem cell biology, differentiation control, the use of stem cells in regenerative medicine, reproduction and development, key areas to ensure fetal and maternal health as well as healthy ageing.

Planned Impact

This project will have substantial impact on many levels, it will benefit the immediate and the wider research community, the clinical areas of obstetrics & gynaecology and child health, it will advance our understanding of requirements for healthy ageing, open up new avenues for regenerative medicine as well as systems biology and biological modelling approaches, and through all this will benefit the public as a whole. The research areas directly affected by our work, nationally and internationally, include stem cell biology, developmental biology and reproductive medicine, obstetrics & gynaecology, cancer biology, systems biology and mathematical modelling, biochemistry as well as cell biology. The results will advance our understanding, and refine modelling, of stem cell behaviour and differentiation potential which is crucial for the therapeutic use in regenerative medicine. They will enable the generation of comprehensive integrated models of genome and chromatin regulation that determine the developmental potency of a (stem) cell which will benefit a wide research community. Our insights will have direct impact on our understanding of mechanisms that underlie ageing and the maintenance of health, and thereby on improving the quality of life. The immediate impact of this study is on advancing our understanding of early development, placentation and healthy pregnancy. Through fetal programming processes, these areas are also of major importance for health in adult life. In the wider field of Obstetrics & Gynaecology they will lead to a better understanding of causes, and in the longer term improved treatments, of pregnancy complications. They will also lead towards refined approaches for the derivation of human trophoblast stem cells whose establishment has not been successful to date. Such stem cells would be most beneficial for research as well as for therapeutic approaches as they would allow, in an unprecedented manner, to study developmental processes early after fertilization where human material is not available. In the longer term they may also prove most valuable for the treatment of common placenta-based pregnancy complications. The wider scientific community will benefit from oral and poster presentations of the ongoing advances of the project at national and international conferences. The results of this work will be published in peer-reviewed open access journals or deposited in open access databases. The integrated genome-wide transcriptional network data will be made available on open-access servers which will enable us and other researchers to expand the dataset in an interactive manner. Potential opportunities for commercial exploitation are provided if the results discover novel mechanisms of stem cell self-renewal and differentiation, maintenance of developmental potency and triggers to avoid stem cell-associated tumourigenic and teraogenic risks. The lay audience will benefit from presentations, press interviews and/or public lectures, and through the social impact of the results of the study. We further actively engage in training the next generation and fostering scientific enthusiasm through visits at schools and School's Day Projects at the Babraham Institute. The project involves in-depth training of the staff involved which will make them highly employable in the academic and commercial research sector, in scientific communication and on scientific/stem cell research advisory boards. The project falls centrally into BBSRC's priority area on stem cell biology. It also has direct impact on the strategic priority area on 'Ageing research: lifelong health and wellbeing', a) by its relevance for normal pregnancy because fetal under-nutrition, e.g. due to a malfunctional placenta, increases the likelihood of cardiovascular disease and diabetes in later life; and b) by its relevance to understand the self-limitation of stem cell proliferation to prevent tumour formation.

Publications

10 25 50
 
Description Stem cells are cells that have the potential to reproduce themselves ('self-renew') as well as to form various terminally
differentiated cell types of the body. For this reason stem cells are of high interest for biomedical research and
regenerative medicine. We are particularly interested in stem cells that contribute exclusively to tissues of the placenta
(so-called trophoblast stem cells). These stem cells are of enormous value to study developmental processes at the
earliest stages of pregnancy where biological material is not available from humans. In the longer term they may also
prove to be valuable for the treatment of pregnancy disorders many of which are based on a defective function of the
placenta, i.e. the organ that mediates all nutrition and oxygen supply to the growing baby.
In this research project, we have investigated the precise pathways that regulate the stem cell state of this unique type of cell. We identified molecular elements that are shared with stem cells specific to the embryo proper, and other that make these placenta-committed stem cells unique. This work, that was carried out in the mouse model, will be instrumental to decipher the conditions required to isolate such a stem cell population from the human placenta.
The work increased research capacity by training several members of staff, in particular the postdoctoral researcher involved, in high-level scientific techniques, project design, data interpretation, writing and presentation skills, as well as personnel management, teaching/training and preparing towards an independent research career as group leader.
Exploitation Route We are taking our insights forward to identify, isolate and hopefully propagate human trophoblast stem cells. These cells would be of fundamental value to gain insights into the developmental processes that occur very early after fertilisation, i.e. during a time window when pregnancy progression (or defects therein) cannot be assessed in humans.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.trophoblast.cam.ac.uk
 
Description The key findings of this research project have been instrumental for our understanding of how the developmental potential of early embryo-derived stem cells is regulated and underpinned by epigenetic mechanisms. Specifically, we have provided milestones how transcription factors intersect with the epigenome by identifying their protein binding partners. We have also established the networks generated by these transcription factors, how they maintain the stem cell state in the trophoblast compartment and how they drive differentiation towards specific placental cell types. We have published important results of this study in various research papers. We will use our insights to drive forward attempts to translate our findings into the early human placenta, with the aim to isolate and derive a trophoblast stem cell population that would be of enormous value for basic research as well as for biomedical translational and drug screening approaches.
First Year Of Impact 2014
Sector Education,Healthcare
Impact Types Societal,Economic

 
Description Research Advisory Board, Wellbeing of Women
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact I served as member of the scientific advisory committee to the charity Wellbeing of Women, which reviews and makes decisions on grant funding in the general field of reproductive medicine. Some of the work funded through WoW has led to improvements in health care, novel diagnostic methods, etc.
 
Description Bayer Grants4Targets
Amount € 20,000 (EUR)
Organisation Bayer 
Department Bayer HealthCare
Sector Private
Country Germany
Start 03/2013 
End 02/2014
 
Description MRC iCASE training award
Amount £111,236 (GBP)
Funding ID MR/M017427/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 09/2020
 
Description Next Generation Fellowship for postdoctoral researcher Dr Paulina Latos
Amount £167,000 (GBP)
Organisation University of Cambridge 
Department Centre for Trophoblast Research
Sector Academic/University
Country United Kingdom
Start 09/2013 
End 08/2016
 
Description Wellcome Trust Strategic Award
Amount £254,000 (GBP)
Funding ID WT100160MA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2013 
End 03/2018
 
Title iTSCs 
Description Novel method that allows for the generation of trophoblast stem cells (TSCs) from fibroblasts to study development and differentiation of the placenta lineage. 
Type Of Material Cell line 
Year Produced 2015 
Provided To Others? Yes  
Impact This method makes the establishment of TSCs tremendously easier, and does not require the isolation of multiple rounds of blastocysts from time-mated females (mice). This in turn reduced the number of required animals. 
 
Title ChIP-seq profiles for numerous transcription factors in TSCs (E-MTAB-3565) 
Description Chromatin binding profiles for Esrrb, Tfap2c, Lsd1, Nr0b1 and others in TSCs 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? No  
Impact Datasets were deposited in publicly available databases. Results helped identify the transcriptional networks operational in trophoblast stem cells, the precursors of the placenta. These insights will be immensely useful for other researchers in the field. 
 
Title DMDD 
Description As part of the Wellcome Trust funded Strategic Award "Deciphering the Mechanisms of Developmental Diseases", we have generated a database in which we collect phenoytping data of the mouse placenta at various gestational stages. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact Broad survey of embryonic lethal gene mutations for defects in embryonic and placental development. Efforts will lead, for the first time, to a global overview of the number and identity of genes, and the molecular pathways they regulate, involved in the development of the placenta as an essential organ ensuring reproductive success and health of the baby. 
URL http://www.nimr.mrc.ac.uk/news/deciphering-the-mechanisms-of-developmental-disorders-progress-report...
 
Title Elf5 and Esrrb protein interactomes in TSCs (PXD002183) 
Description IP->MS/MS datasets of interaction partners of the key transcription factors Esrrb and Elf5 in trophoblast stem cells (TSCs). 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? No  
Impact Data deposited in publicly accessible databases. Results are instrumental to gain a detailed understanding of how TSCs operate, which will be important to move the field of reproductive biology and pregnancy (normal and pathological) forward. 
URL http://www.ebi.ac.uk/pride/archive/
 
Description Bioinformatics analysis of ChIP-seq and RNA-seq data 
Organisation EMBL European Bioinformatics Institute (EMBL - EBI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided extensive high-throughput sequencing datasets for analysis.
Collaborator Contribution Expert analysis of ChIP-seq data and data mining of existing datasets
Impact Multi-discipliniary, involving mathematical biology and bioinformatics. Joint manuscript submitted October 2014
Start Year 2014
 
Description Bioinformatics analysis of ChIP-seq and RNA-seq data 
Organisation University of Toronto
Country Canada 
Sector Academic/University 
PI Contribution Provided large-scala datasets for analysis.
Collaborator Contribution Analysed the data.
Impact Latos P.A., Sienerth A.R., Murray A., Senner C.E., Muto M., Ikawa M., Oxley D., Burge S., Cox B. and Hemberger M. (2015). Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks. Genes & Development, 29: 2435-2448.
Start Year 2014
 
Description Epigenetic memory in ES-to-TS cell reprogramming 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution Collaboration with Dr Simon Cook on the impact of Ras-Raf-Erk signalling on ES cell reprogramming towards trophoblast. Designed overall project outline and performed experimental analyses.
Collaborator Contribution Provided plasmid constructs and antibody resources. Provided help and advice on specific Western blotting techniques. Provided ongoing scientific advice.
Impact Cambuli F., Murray A., Dean, W., Dudzinska D., Krueger F., Andrews S., Senner C.E., Cook S. J. and Hemberger M. (2014). Epigenetic memory of the first cell fate decision prevents complete ES cell reprogramming into trophoblast. Nat. Commun., 5: 5538.
Start Year 2012
 
Description Reprogramming of MEFs towards iTSCs and establishment of defined TSC culture conditions 
Organisation University of Bonn
Country Germany 
Sector Academic/University 
PI Contribution Genome-wide DNA methylation analysis of various clones and reprogrammed cells. Bioinformatic analysis of these data. Figures for manuscripts.
Collaborator Contribution Provided DNA for the above analysis. Performed other experiments to bring this project to conclusion.
Impact Kubaczka C., Senner C.E., Araúzo-Bravo M.J., Sharma N., Kuckenberg P., Becker A., Zimmer A., Brüstle O., Peitz M., Hemberger M. and Schorle H. (2014). Derivation and maintenance of murine trophoblast stem cells under defined conditions. Stem Cell Reports, 2: 232-242. Kubaczka C., Senner C.E., Cierlitza M., Araúzo-Bravo M.J., Kuckenberg P., Peitz M., Hemberger M. and Schorle H. (2015). Direct induction of trophoblast stem cells from murine fibroblasts. Cell Stem Cells, 17: 557-568.
Start Year 2012
 
Description Trophoblast-specific overexpression 
Organisation University of Tokyo
Country Japan 
Sector Academic/University 
PI Contribution Collaboration on the establishment of a key mouse mode, which Prof Ikawa has pioneered
Start Year 2012
 
Description AM, participation in International Society for Stem Cell Research Meeting, Vancouver, Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact The PhD student who attended this conference was selected for poster presentation at this prestigious international meeting.

Widening of scientific horizon, valuable experience in presentation skills, scientific interaction, establishing links with the scientific community world-wide
Year(s) Of Engagement Activity 2014
 
Description Conference The stem cell niche (Denmark) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact This grant started in September 2011, with the appointment of a postdoctoral scientist (Dr Paulina Latos) supported by this grant. Dr L submitted an abstract reporting her first findings to a prestigious meeting, "The stem cell niche" held in Copenhagen in June 2012, which was accepted for poster presentation. It should be noted that this was a competitive selection as most of the meeting costs are waived for selected participants. This is a first great achievement for Paulina and the output of the grant investment.

raised profile of postdoctoral researcher in front of an international audience
Year(s) Of Engagement Activity 2011
 
Description Member, Board of Managers at Centre for Trophoblast Research 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Supporters
Results and Impact As member of the Board of Managers at the CTR in Cambridge we make decisions on the award of PhD scholarships, postdoctoral fellowships and direct the general focal points of local research activity in the field of placental biology.

PhD students admitted to the University of Cambridge; postdoctoral researchers' career path enhanced
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Royal Society Partnering Award - CS + LW, NM, SC 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Successful application for Royal Society Partnering Award. Poster 6 selected 6th form students over the course of 1 week and conducted in-depth research project. Data analysis followed at the pupils' school directly.
Year(s) Of Engagement Activity 2016,2017
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact presented basic research topic (intrauterine development and essential function of placenta) to primary school children

awareness and excitement of young people for science
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description School's Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact provided basic explanation of early development and the importance of the placenta for growth of a baby; hands-on experience for students in staining and microscopic analysis of a mouse placenta

rose awareness of research activities and importance of basic research to understand common pregnancy-associated diseases
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Schools Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Project design, scientific background induction and practical supervision for 6th form students as well as teachers during Annual Schools Day.
Year(s) Of Engagement Activity 2015,2016,2017