Elucidating novel pluripotency signalling networks

Lead Research Organisation: University of Dundee
Department Name: School of Life Sciences

Abstract

I will investigate the proteins that control stem cell behaviour.

Stem cells are a very important and interesting cell type, as they can either divide into a larger population of stem cells ("self-renew"), or develop into one of the many types of tissues and organs in the human body ("differentiate"). The balance between these alternative cell fates is fundamental to human growth and development, and this process often go awry in human disease. In addition, it is possible for researchers to instruct stem cells to develop into adult tissues and organs in the laboratory. Therefore, by fully understanding stem cell behaviour, we may be able to both treat diseases and manipulate stem cells to generate replacement tissues and organs for patients; for example, by using stem cells to produce heart muscle to repair a damaged heart following a heart attack.

Whether stem cells self-renew or differentiate is controlled by signals from their immediate environment. However, the ways in which stem cells interpret these signals is poorly understood. We therefore need to identify the lines of communication used by stem cells to interpret environmental signals leading to a change in stem cell behaviour. Knowledge of the communication lines that tell a stem cell how to behave will potentially allow us to re-wire these communication systems so that we can either keep stem cells growing indefinitely, or instruct them to develop into adult tissues and organs.

One of the most important components of cell communication systems are proteins known as kinases, which play an important role in determining stem cell behaviour. Kinases can be readily blocked using chemicals, which disrupts the lines of communication. This is a relatively simple way to rewire stem cell communication systems so that the stem cells behave in a particular way. Therefore, this research project aims to uncover the kinases that control stem cell behaviour, knowledge which potentially will be exploited to form cells of a particular tissue or organ using chemicals.

In my recent research we have found a kinase known as ERK5, which is used by stem cells to stop self-renewing and start development towards adult tissues and organs. I aim to investigate how ERK5 is connected to ESC communication systems, and whether we can use chemicals to alter ERK5 communication patterns to modify stem cell behaviour. I also aim to identify other kinases that are involved in other important ESC communication systems, and again use chemicals to shut down these systems and artificially change stem cell behaviour in the laboratory.

Technical Summary

Pluripotent Embryonic Stem Cells (ESCs) have the capacity to differentiate into all cell types in the adult body, and their application for the purposes of regenerative medicine and disease modelling has shown significant promise in recent years. However, it remains a challenge to harness the developmental capacity of ESCs, thereby capturing distinct pluripotent and differentiated states for use in regenerative therapeutics. A highly promising novel approach is to identify components of the phosphorylation machinery that control pluripotency and differentiation. Phosphorylation networks ultimately modulate the function of key regulatory targets such as transcriptional circuits to direct developmental potential. However, the kinases involved in these regulatory systems have not been extensively explored.

Here, I propose to uncover fundamental molecular bases of pluripotency regulation and identify strategies to manipulate key signalling components using small molecules. The primary aim of this project is to identify novel kinases and mechanisms by which phosphorylation controls gene expression to modulate the developmental potential of ESCs. Preliminary data from my lab suggests that the ERK5 kinase signalling pathway is a key regulator of pluripotency, and I will explore the molecular mechanisms underpinning this regulation. I will also exploit an inter-disciplinary combination of cutting-edge chemical biology, gene editing technology and phenotype-based analysis to identify and specifically perturb novel kinases that modulate ESC pluripotency. My extensive preliminary data, including identification of the ERK5 pathway and validation of a high-throughput screen for kinase inhibitors that modulate pluripotent ESC states, demonstrates the feasibility of the proposal. This research will generate new opportunities for chemical manipulation of ESC phosphorylation networks, allowing distinct developmental fates to be synthetically programmed in the laboratory.

Planned Impact

This project aims to identify novel kinases and signalling pathways that control ESC pluripotency and differentiation. Therefore, this research will benefit:

1. The academic researchers directly involved in the project, by developing their research skills and experience through this varied, ambitious and multi-disciplinary research. The researchers involved will also benefit by acquiring further professional and transferable skills, in particular the PDRA, who will be assisted and encouraged to develop public engagement and communication skills in a variety of ways.

2. The worldwide academic community, including researchers interested in signal transduction, protein kinases, ESC biology including human ESCs, developmental pathways and cellular programming for regenerative medicine/disease modelling. More broadly, my research will also benefit scientists and clinicians who work in application and translation of Stem Cell technologies. These communities will immediately gain new ideas and scientific knowledge from our data, which in future will lead to development of novel methodologies and protocols to improve our basic understanding and medical application of Pluripotent Stem Cells. In addition, my laboratory will generate and make available many tools and reagents to study kinases identified in this research (including antibodies, cDNA constructs and mutants, kinase inhibitors and knock-out ESC lines) to facilitate research projects in other laboratories.

3. Commercial private companies aiming to develop effective platforms for reprogramming, culturing and differentiating Pluripotent Stem Cells for regenerative applications and disease modelling. In particular, we envision that our findings will be primarily exploited by MRC-PPU partner companies and other interested parties to elaborate novel protocols and methods to improve the feasibility and success of Stem Cell approaches for use in commercially-viable disease treatments. Improved Pluripotent Stem Cell culture protocols using kinase inhibitors is a key outcome which may be realised rapidly, whilst long-term applications will include elaboration of novel protocols and procedures, based on manipulation of kinase signalling, to facilitate generation of differentiated tissues and lineages for regenerative therapies and disease modelling. Finally, private companies with access to small molecule programmes and sophisticated chemical biology will potentially identify and develop drugs based on kinase inhibitors identified in our research, which may in future be used as drugs to treat disease.

4. The general public will benefit from this research through increased awareness and understanding of science, particularly in my field of Stem Cell biology. I will be an active participant in public outreach, introducing simple concepts of Stem Cells and their potential uses in treatments of disease, and publicising exciting findings from my laboratory to those beyond the academic and pharmaceutical communities. The MRC-PPU is committed to a number of outreach forums, which I will utilise to deliver effective public outreach. In the longer-term, this research aims to contribute to improvements in public health and welfare by providing a more detailed understanding of pluripotency and differentiation, and mechanisms by which these can be modulated, to facilitate development of effective regenerative therapeutics and disease modelling strategies to improve treatment of a variety of diseases.

Publications

10 25 50
 
Title EMBO Reports cover image 
Description A fluorescent image of Embryonic Stem Cells in culture generated by our laboratory was selected by EMBO Reports journal as the cover for Volume 18 Issue 7, 3 July 2017 
Type Of Art Image 
Year Produced 2017 
Impact N/A 
URL http://onlinelibrary.wiley.com/doi/10.1002/embr.v18.7/issuetoc
 
Description Tenovus Research Grant
Amount £15,000 (GBP)
Organisation Tenovus 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2016
 
Description Janssen 
Organisation Johnson & Johnson
Department Janssen Pharmaceutica
Country Global 
Sector Private 
PI Contribution My research team conducted phenotypic screening of small molecule kinase inhibitor compounds from Janssen to identify those which modulate Embryonic Stem Cell pluripotency
Collaborator Contribution Janssen's Molecular Informatics department provided my laboratory with a collection of ~450 high value potent and selective small molecule kinase inhibitors for phenotypic screening
Impact Phenotyping of these inhibitors allowed my laboratory to identify novel kinases involved in regulation of Embryonic Stem Cell pluripotency
Start Year 2014
 
Description Nathanael Gray Erk5 inhibitors 
Organisation Dana-Farber Cancer Institute
Country United States 
Sector Hospitals 
PI Contribution My research team utilised small molecule kinase inhibitors and genome editing technology to identify a novel role for the Erk5 kinase in regulation of Embryonic Stem Cell pluripotency and differentiation
Collaborator Contribution Nathanael Gray's laboratory contributed state of the art potent and selective small molecule Erk5 kinase inhibitors
Impact Publication: Erk5 Is a Key Regulator of Naive-Primed Transition and Embryonic Stem Cell Identity. Williams CA, Fernandez-Alonso R, Wang J, Toth R, Gray NS, Findlay GM. Cell Rep. 2016 Aug 16;16(7):1820-8. PMID: 27498864
Start Year 2015
 
Description Prof. Alessio Ciulli (BCDD, SLS Dundee) - applications of Brd4/BET Proteolysis Targeting Chimera (PROTAC) small molecule degraders 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution My team employed PROTAC small molecule degraders to elucidate novel functions of Brd4/BET bromodomain proteins in Embryonic Stem Cells
Collaborator Contribution Prof. Ciulli provided us with cutting edge PROTAC molecules his lab developed to specifically degrade the Brd4 BET bromodomain protein
Impact Publication: Brd4-Brd2 isoform switching coordinates pluripotent exit and Smad2-dependent lineage specification. Fernandez-Alonso R, Davidson L, Hukelmann J, Zengerle M, Prescott AR, Lamond A, Ciulli A, Sapkota GP, Findlay GM. EMBO Rep. 2017 Jul;18(7):1108-1122. PMID: 28588073
Start Year 2015
 
Description Prof. Angus Lamond (GRE, SLS Dundee) - absolute quantitative total proteome analysis of Embryonic Stem Cells 
Organisation University of Dundee
Country United Kingdom 
Sector Academic/University 
PI Contribution My team contributed Embryonic Stem Cell expertise and samples.
Collaborator Contribution Prof. Lamond's team contributed expertise in mass spectrometry for absolute quantitative total proteome analysis of Embryonic Stem Cells.
Impact Publication: Brd4-Brd2 isoform switching coordinates pluripotent exit and Smad2-dependent lineage specification. Fernandez-Alonso R, Davidson L, Hukelmann J, Zengerle M, Prescott AR, Lamond A, Ciulli A, Sapkota GP, Findlay GM. EMBO Rep. 2017 Jul;18(7):1108-1122. PMID: 28588073
Start Year 2015
 
Description Wolf Reik (Babraham Institute, UK) - Regulation of DNA methylation and germ cell development by signalling pathways 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution We are identifying new mechanisms by which protein kinase signaling regulates the DNA methylation machinery. We use mass-spectrometry to identify phosphorylation sites on UHRF1, a key E3 ubiquitin ligase that controls DNA methyltransferase.
Collaborator Contribution The Reik lab is investigating how kinases and E3 ubiquitin ligases that we are studying regulate germ cell development using a stem cell model
Impact N/A
Start Year 2017
 
Description "Courier & Advertiser" newspaper article, 29th Aug 2018 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact A press released issued about a recent major fellowship award and my lab's research formed the basis of an article in the regional "Courier & Advertiser" newspaper. I have since been approached by members of the public requesting more information about our research.
Year(s) Of Engagement Activity 2018
 
Description Art & Science Collaboration 
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 Postgraduate students
Results and Impact In an effort to try public engagement in a different way, the PPU will be inviting some artists shortly. As a reminder, we'll be having 6 artists from the Medical Science Illustrators course at The Duncan and Jordanstone College of Art & Design visited The MRC Protein Phosphorylation and Ubiquitylation Unit.

The goal of this effort was a few-fold:
1. To identify points in investigators' own research that might be conveyed in a different way to the general public as well as an artist who can assist them;
2. To identify a project for the artists who may wish to learn more about the science going on so close to home
3. To create a final piece which (ideally) may become part of an installation in the Discovery Centre at The University of Dundee to convey science to new audiences
Year(s) Of Engagement Activity 2015,2016
 
Description Dundee Science Festival - The Wonder of the Brain 2014 
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 As a part of the one of the events organized by the MRC-PPU - The Wonders of the Brain: Big and Small! - visitors were encouraged to partake in a trivia challenge, all as they journeyed through the exhibit where they learned about the brain, viewed models from different species, and evaluated example MRIs and illustrations that highlighted changes that occur during disease. The games that were a part of this exhibit were also a great hit as children matched neurotransmitter 'vesicles' with the proper receptor, made neurons out pipe-cleaner, made their very own thinking caps and 'trained their brain' using memory and motor games.

Students and children were encouraged children to learn about the brain, participate in a wide array of games and make models of individual neuronal cells. Besides coming away with a greater appreciation for the brain as an organ, all visitors expressed a positive experience.
Year(s) Of Engagement Activity 2014
 
Description Dundee Science Festival, Street Food, Nov 2017. Francisco Bustos and Rosalia Fernandez-Alonso (PDFs) presented our lab's research at a public tasting of local produce in SLS 
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 Francisco Bustos and Rosalia Fernandez-Alonso (PDFs) presented our lab's research to 100 members of the general public at "Street Food", a combined exhibition of local scientific research and produce hosted as part of the Dundee Science Festival by School of Life Sciences, University of Dundee.
Year(s) Of Engagement Activity 2017
 
Description Hosting Baldragon students in the lab 
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 2 senior biology pupils from Baldragon Academy visited my lab for a day, which stimulated their interest in pursuing a career in scientific research.
Year(s) Of Engagement Activity 2015
 
Description Interview with STV news, 29th Aug 2018 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact I gave an interview to Scottish Television News about a recent major fellowship award and my lab's research. I have since been approached by members of the public requesting more information about our research.
Year(s) Of Engagement Activity 2018
 
Description Interview with That's TV Scotland, Aug 30th 2018 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact I gave an interview to That's TV Scotland news channel about a recent major fellowship award and my lab's research. I have since been approached by members of the public requesting more information about our research.
Year(s) Of Engagement Activity 2018
 
Description Interview with local radio station 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact On the day of publication of Williams et al, 2016 (Cell Rep), I conducted a short radio interview with local radio station Wave102. This interview was broadcast in news bulletins throughout the day across the local region (Tayside and Fife)
Year(s) Of Engagement Activity 2016
 
Description Lucia Rodriguez-Garcia internship, 8-12th Jan 2018 
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 Undergraduate students
Results and Impact Lucia Rodriguez-Garcia, an undergraduate student at the University of Edinburgh, conducted a one week research internship in my laboratory. Afterwards, Lucia reported an increased interest in stem cell research as a potential career route.
Year(s) Of Engagement Activity 2018
 
Description MRC-PPU Collaboration with Baldragon Academy 
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 The Medical Research Council's Protein Phosphorylation and Ubiquitylation Unit (MRCPPU), part of The University of Dundee, has prioritized public engagement in an effort to engage the general public and ensure that the research activities and breakthroughs are communicated to the community. Of equal importance in these communication efforts is educational outreach to students within the Dundee community.

Thus, during session 2014 -2015 and 2015-2016 school year, the MRC-PPU will partner with a local secondary school -- Baldragon Academy (BA). Teachers in BA's Science Department will collaborate with scientists at the MRC-PPU in an educational outreach effort (see Appendix 1). The purpose of this project is to increase interest and engagement in science and related careers. It will be starting in August 2014 with the S1 pupils.

Scientists from the unit will be working with the pupils on a monthly basis at the school during their science classes and will be providing them with opportunities to take part in various science experiments and demonstrations (aligned with Scotland's Curriculum for Excellence). The scientists are leaders in their field of research and as such come from all over the world. They are currently based in Dundee."


Thus far, student have been very enthusiastic about the labs and very receptive to the volunteers. They have asked a multitude of quesitons and have even asked volunteers back to visit.
Year(s) Of Engagement Activity 2014,2015,2016
 
Description Opinion for The Scotsman Newspaper, 7th Dec 2017 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Greg Findlay (PI) provided expert opinion in "The Scotsman" national newspaper for an article about recent stem cell research 'Stem cells "will enable us to live to 200"'
Year(s) Of Engagement Activity 2017
 
Description Presentation for SOAPBOX SCIENCE, The Mound, Edinburgh, 2nd June 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Helen Brown, a PhD student in the Findlay, got on her soapbox to share the truth about stem cell manipulation and its novel impact in medicine.

Soapbox Science is a novel public outreach platform promoting women scientists and the science they do. Following the format of London Hyde Park's Speaker's Corner, which is historically an arena for public debate, Soapbox Science, wants to make sure that everyone has the opportunity to enjoy, learn from, heckle, question, probe, interact with and be inspired by some of the UK's leading scientists. She joined researchers from across Scotland, as they share their passion for science with the public from the historic Mound, next to the National Galleries in Edinburgh.

Helen said, "I am delighted to get on my Soapbox this weekend. Soapbox Science has captured the imaginations of hundreds over the last three years and I cannot wait for my opportunity to get up there and share the story of stem cells.

"Soapbox Science is doing a great job of increasing the visibility of women in science. By inviting female researchers to talk in public at a historically male-dominated venue (the Mound), it aims to address the fact that women tend not to pursue science to a high level.

"This weekend is a fantastic opportunity for anyone thinking of taking science further."
Year(s) Of Engagement Activity 2018
URL http://soapboxscience.org/
 
Description Providing expert opinion for the "Entrepreneur First" investment scheme, 24th May 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact I had a discussion with Dr. Steven Renwick, an entrepreneur based in Berlin, Germany, who is part of the "Entrepreneur First" investment scheme. This scheme aims to fund and develop a broad range of new business concepts, including in the area of scientific research. Dr Renwick is a molecular biologist by training, and is pursuing entrepreneurship opportunities based on non-consensus ideas in scientific publishing. I was identified by Dr. Renwick as an expert in the area of scientific research, and provided my opinion on the areas of scientific publishing in which new business opportunities might arise.
Year(s) Of Engagement Activity 2018
 
Description School visit (Brechin) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 20 pupils studying biology attended a stem cell workshop I presented at Brechin High School, with questions and discussion afterwards, and the school reported increased interest in biology and stem cells afterwards.
Year(s) Of Engagement Activity 2014
 
Description Stem Cell Ethics visit 
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 60 pupils studying S4 biology (14-15 years old) attended a Stem Cell Ethics workshop here in the School of Life Sciences, University of Dundee. Colleagues and I presented ethical questions related to Stem Cells, which was followed by ethical debate and discussion. The school reported increased interest from students in continuing their studies in biology and stem cells afterwards.
Year(s) Of Engagement Activity 2016
 
Description Stem Cell Ethics workshop 
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 Greg Findlay (PI), Charles Williams (PhD) and Francisco Bustos (PDF) led stem cell ethics discussion groups with high school pupils, which sparked increased interest and change in perceptions about stem cell research and applications.
Year(s) Of Engagement Activity 2016
 
Description Street Food festival, University of Dundee, 18th Oct 2018. Members of the Findlay lab presented our lab's research to the public in conjunction with tasting of local produce 
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 In a link up with local coffee roastery "Sacred Grounds", members of the Findlay lab presented our research into differentiation of stem cells into cardiomyocytes (heart muscle). Attendees of the Street Food event were given a tour of the lab, and shown experiments in which stem cells were differentiated into heart muscle cells. Lab members also measured heart rate of attendees before and after sampling freshly brewed Sacred Grounds coffee, demonstrating the effect of caffeine on the heart.
Year(s) Of Engagement Activity 2018
URL https://www.dundee.ac.uk/festival-future/programme/2018/18-10-18-street-food-.php
 
Description University of St Andrews Biological Society, Helen Brown talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Helen Brown (PhD student) gave a talk about her research on stem cells to an undergraduate audience at the University of St Andrews Biological Society with discussion afterwards. The audience expressed an increased interest in stem cell research.
Year(s) Of Engagement Activity 2017
 
Description UoD contact magazine article 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact The UoD press release conceived an article on Stem Cell Research to be published in the widely distributed UoD magazine "Contact". In this article, Greg Findlay discussed his own research, and Stem Cell Research being conducted in Dundee and beyond.
Year(s) Of Engagement Activity 2016
 
Description Williams et al newspaper articles 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact On the day of publication of Williams et al, 2016 (Cell Rep), a press release was issued in conjunction with the University of Dundee press office. Material from this formed the basis of two newspaper articles (one including photograph) published in regional newspapers the Courier and Advertiser (Thursday 18th August 2016) and the Evening Telegraph (Wednesday 17th August 2016).
Year(s) Of Engagement Activity 2016