Investigating and manipulating the changing display of glycosaminoglycan epitopes during ES cell differentiation

Lead Research Organisation: University of Manchester
Department Name: Materials

Abstract

All of the cells which make up the tissues of the body (e.g. the skin, liver, brain and blood) are surrounded by a layer of sugars which coat the cells. This sugar-coating is different for different cell types so, for example, a skin cell will have a type of sugar coat which is not the same as that of a liver cell. The sugars help the cells to know what type of cell they are and to respond to the other cells which surround them as well as to the chemical messages that pass between cells. Glycosaminoglycans are a special type of sugar which are part of the cell coat and also fill in the spaces between cells within tissues. This family of sugars have many unique properties which include the ability to prevent blood clotting (heparin), to enable joints to move freely (chondroitin sulphate) and to allow eggs to mature during the female reproductive cycle (hyaluronan). At present, the way in which cells make these sugars is not well understood. From the little we do know, we believe that isolated fragments of these sugars could be used to instruct cells to behave in particular ways, or that we might be able to force cells to make one particular type of sugar and not another, thereby influencing the way in which that cell grows and interacts with other cells. In this research plan, we will use embryonic stem (ES) cells to help understand how different cells make different sugar types and to test out our theories as to how sugars can influence cell behaviour. We have chosen ES cells for two main reasons. These cells are unusual in that they can be grown easily in the lab and can change from being one cell type (an ES cell) to another (e.g. a nerve cell). This will help us to understand how the sugars made by the cells change during this process. An additional benefit of using ES cells is that our research might suggest how sugars can be used to help ES cells grow in the lab or how they can be instructed to become cell types which could be of use in human therapies e.g. nerve cells, heart muscle cells or blood cells. Although cells made from ES cells are still a considerable time away from being used in people, research such as ours helps move towards this goal.

Technical Summary

Glycosaminoglycans (GAGs) are essential cofactors for many of the signalling molecules which regulate embryonic stem (ES) cell pluripotency and differentiation. Whereas many groups are investigating the protein components of these signalling complexes, the carbohydrate fraction is less well understood and therefore remains an under-appreciated factor in the design of fully-defined ES cell culture systems or methods to direct differentiation. This project aims to extend our current limited knowledge of the function of specific GAG epitopes in mouse and human ES cells, and to use this information to manipulate ES cell differentiation. Recent evidence suggests that increasing sulphation within GAGs displayed on ES cells during early differentiation is essential for their efficient response to signalling factors. A panel of phage display-derived ScFv antibodies, previously used to detail specific patterns within heparan sulphate (HS) chains in mouse ES cells, will be expanded to include a study of human ES cell differentiation as well as to include ScFvs which recognise chondroitin and dermatan sulphate (CS/DS) epitopes. Focusing on neural differentiation, epitopes within GAG chains found to be implicated in the ability of the ES cells to respond to pro-differentiation signals will be targeted to directly assess the role of GAG biosynthetic enzymes and isolated GAG oligosaccharides in influencing cell behaviour.

Publications

10 25 50
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Ayerst BI (2017) The Good the Bad and the Ugly of Glycosaminoglycans in Tissue Engineering Applications. in Pharmaceuticals (Basel, Switzerland)

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Giangreco A (2014) The biochemical determinants of tissue regeneration. in Biochemical Society transactions

 
Description Participation in the International Proteoglycan Training Course
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description White paper
Geographic Reach National 
Policy Influence Type Participation in a national consultation
URL http://ibcarb.com/wp-content/uploads/White-Paper-v7.pdf
 
Description Biochemical Society Workshop
Amount £30,000 (GBP)
Organisation Biochemical Society 
Sector Learned Society
Country United Kingdom
Start 04/2017 
End 05/2017
 
Description EPSRC EP/H046070/1 Stem cell fractionation using interactions with artificial matrices
Amount £2,175,436 (GBP)
Funding ID EP/H046070/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 09/2010 
End 09/2014
 
Description EPSRC KTA
Amount £75,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 09/2011 
End 08/2012
 
Description EPSRC Next Generation Biomaterials Studentship
Amount £5,400,000 (GBP)
Funding ID EP/N006615/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description EPSRC RegenMed DTP
Amount £88,000 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 09/2016 
End 08/2020
 
Description FAPESP University of Nottingham, University of Birmingham collaboration scheme
Amount £40,000 (GBP)
Organisation São Paulo Research Foundation (FAPESP) 
Sector Public
Country Brazil
Start 01/2018 
End 06/2019
 
Description FP7
Amount £500,000 (GBP)
Organisation European Commission 
Department Seventh Framework Programme (FP7)
Sector Public
Country European Union (EU)
Start 01/2010 
End 09/2011
 
Description MCR studentship
Amount £72,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 08/2020
 
Description MRC/BHF joint Research Grant
Amount £741,792 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 09/2011 
End 08/2013
 
Description Research Grant/Human Frontiers Scientific Programme
Amount £562,650 (GBP)
Organisation Human Frontier Science Program (HFSP) 
Sector Charity/Non Profit
Country France
Start 09/2008 
End 08/2011
 
Description The Leverhulme Trust
Amount £189,360 (GBP)
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 09/2011 
End 08/2012
 
Description The Leverhulme Trust Visiting Professorship
Amount £18,720 (GBP)
Funding ID VP-2016-014 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 02/2017 
End 07/2017
 
Description Tools and Resorces Fund
Amount £137,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2009 
End 08/2010
 
Description University of Nottingham Regenerative Medicine RPA
Amount £10,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 09/2017 
End 03/2018
 
Title Combination of compositional and pattern-based glycosaminoglycan detection 
Description We have developed methods to allow the combined analysis of cell-surface glycosaminoglycans using sensitive compositional analysis (fluroescent tagging and disaccharide analysis) and antibody-based analysis of the pattern-based carbohydrate epitopes displayed by the chains. 
Type Of Material Data analysis technique 
Year Produced 2010 
Provided To Others? Yes  
Impact We have trained other groups in these techniques and provide on-going support for their analyses. We are often named as joint-authors on publications or acknowledged for our support. These analyses help significantly with defining the biological role of these complex sugars. 
 
Title GAGs to drive differentiation of progenitor cells 
Description We have completed our initial studies to define and characterise the role of heparan sulphate-type oligosaccharides in driving the formation of neural progenitor cells from embryonic stem cells. We have characterised the signalling pathways influenced by these oligosaccharides and can now start to apply these to human progenitor cell differentiation. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact We are working with other groups to use these oligosaccharides to drive differentiation in multiple different progenitor cell assays. We are also working with other group developing biomaterial scaffolds on which to display these oligosaccharides. 
 
Description BIOSCENT 
Organisation University of Manchester
Department School of Materials Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide expertise in glycosaminoglycans which are used to functionalise hydrogels to optimise cell response.
Collaborator Contribution This collaboration provides us with access to a large international grouping of researchers and clinicians. The group include cardiac surgeons, basic scientists, biomaterials engineers and compliance experts focused on developing novel functionalised biomaterials for cardiovascular repair.
Impact EU FP7 grant awarded in 2009. The collaboration is interdisciplinary involving clinicians, basic scientists and biomaterials engineers.
Start Year 2009
 
Description HFSP 
Organisation University of Strathclyde
Department Department of Pure and Applied Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We bring our glycosaminoglycan and stem cell expertise to this collaboration. We also have experience of other hydrogel systems which is useful in improving hydrogel design.
Collaborator Contribution This collaboration is with two biomaterial chemists with outstanding international reputations. We are working to develop hydrogels which can be functionalised with glycosamionglycans to support and direct stem cell culture.
Impact We reveived funding from the Human Frontiers Scientific Programme to support this collaboration. I have also co-supervised a PhD student working on a project related to this programme with Prof. Ulijn.
Start Year 2008
 
Description Investigating the vascular progenitor cell niche 
Organisation Wellcome Trust
Department Wellcome Trust Centre for Cell-Matrix Research
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We contribute expertise in glycosaminoglycan analysis. This includes the structural and functional assays we have developed during the period of the NIRG grant. We also contribute expertise in biomaterial design and testing.
Collaborator Contribution This collaboration allows us to expand our investigations of the role of glycosaminoglycans in regulating stem cell behaviour to the adult stem cell niche. The collaborating team also include experts in animal models as well as clinicians ensuring that there is a high level of clinical relevance to the work.
Impact We were awarded an MRC grant to support this work (G0902170 MRC-BHF Cell-matrix biology of the vascular progenitor cell niche.) which will start in Jan 2011. This collaboration is multi-disciplinary with clinicians, life scientists and bio-engineers working together on the project.
Start Year 2009
 
Description Lena 
Organisation Uppsala University
Country Sweden 
Sector Academic/University 
PI Contribution I have a long standing collaboration with this group. We have worked together to investigate the biosynthetic pathway of glycosaminoglycans. More recently, I have provided my expertise in biomaterials with which to mimic the extracellular matrix and in stem cell biology.
Collaborator Contribution The Uppsala group have world class expertise in glycosaminoglycan structural and functional characterisation as well as the use of animal models.
Impact We have co-published one paper to date. In 2014 I was awarded a Leverhulme Trust fellowship to spend a year in Sweden working with the Uppsala group. During this time we planned out a large EU consortium to apply a new approach to the study of rare disorders of GAG metabolism. We were unsuccesful with an EU H2020 project grant but recenlty resubmitted this as an MSCA ITN.
 
Description Leverhulme 
Organisation University of Manchester
Department School of Materials Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We are providing stem cell expertise and our knowledge of stem cell-matrix interactions. The experince we have gained from working with other biomaterials is also useful for this collaboration.
Collaborator Contribution This collaboration includes a tissue engineer in Manchester and a biomaterials chemist in Glasgow. We are working to better understand the role of mechanotransduction in regulating stem cell behaviour using novel engineered nano-patterned surfaces.
Impact We received funding from the Leverhulme Trust to support this collaboration, the project will start in Jan 2011. This project is interdisciplinary linking chemists with tissue engineers and stem cell biologists.
Start Year 2009
 
Description Marios 
Organisation University of Dundee
Department Centre for Oncology and Molecular Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide expertise in glycosaminoglycans and how these sugars interact with signalling factors to influence stem cell behaviour.
Collaborator Contribution Dr Stavridis has been a long-standing collaborative partner. This collaboration provides us with expertise in the signalling pathways underlying stem cell differentiation.
Impact We have co-published papers with Dr Stavridis.
 
Description Orla Protein Technologies 
Organisation Orla Protein Technnologies
Country United Kingdom 
Sector Private 
PI Contribution We have worked with Orla as part of a 6 month knowledge transfer project to develop novel protein-based detection devices for heparan sulphate.
Collaborator Contribution Orla Protein Technologies are an SME which develop protein-based surfaces engineerd to express matrix-derived motifs. We have been working with Orla to optimise this system for the recognition of heparan sulphate and to display heparan sulphate-binding peptides.
Impact We were awarded an EPSRC-funded knowledge-transfer award to fund this collaboration for six months this was worth ~ £75K, together with in-kind funding from the industrial partner. We are currently applying for a patent to cover the invention. This project is interdisciplinary, combining engineering (surface analysis), protein and sugar biochemistry and medicine (the target here are clinically relevant samples containig heparan sulphate.)
Start Year 2009
 
Description Stem Cell Fractionation 
Organisation University of Manchester
Department Faculty of Engineering and Physical Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide knowledge of the cell-matrix interface and of potenital motifs which may be used to selectively bind specific cell types.
Collaborator Contribution This collaborative group includes experts in microfluidic technologies, matrix biology and stem cells. The group are working to develop a novel technology for the fractionation of stem cell populations based on their adhesion to matrix motifs. This is a novel application for our research.
Impact We received a research grant from EPSRC to fund this collaboration. The project started in September 2010. The project is multi-disciplinary including engineers, life sceintists and materials scientists.
Start Year 2009
 
Description Ward 
Organisation University of Manchester
Department School of Dentistry Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide knowledge of cell-matrix interactions and how these might interact with cell-cell interactions in influencing stem cell behaviour.
Collaborator Contribution Collaboration with Dr Ward's group enables us to explore various aspects of stem cell biology. In particular, we have collaborated on investigating the role of cell-cell interactions. Dr Ward's group also provide technical expertise in human ES cell culture.
Impact I co-supervise a number of PhD and Masters level students with Dr Ward and we have co-published a number of research papers and reviews. We also collaborate on the EPSRC stem cell fractionation project.
 
Description Whitelock 
Organisation The Leverhulme Trust
Country United Kingdom 
Sector Academic/University 
PI Contribution I am hosting a visit by Prof. John Whitelock. While working with our group John is learning how to carry out CRISPR modification of embryonic stem cell lines to model extracellular matrix related disease.
Collaborator Contribution While working with our group John is supporting multiple projects providing knowledge and reagents to investigate the extracellular matrix in natural and synthetic matrices.
Impact The collaboration is multi-disciplinary, covering cell biology, biochemistry and materials science. Outputs so far are primary increased knowledge and reagent development.
Start Year 2017
 
Title heparan sulphate binding proteins 
Description We are developing these reagents with Orla Protein Technologies. We were funded by an EPSRC KTA study and have continued the collaboration with the support of an EPSRC research grant. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Initial development
Year Development Stage Completed 2011
Development Status Under active development/distribution
Impact The development is at too early a stage to give these details yet. 
 
Description BBC Radio Wales 'Science Cafe' Interview 
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 We were interviewed by BBC Wales for their 'Science Cafe' - we talked about the hydrogel technology and how this could be used for tissue engineering or for toxicity screening.

BBC Radio Wales Interview

none so far
Year(s) Of Engagement Activity 2013
 
Description Presentation to an American patient support group for Multiple Osteochondromas 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I attended a meeting organised by the American patient support group for Multiple Osteochondromas. This disease is related to my research work and they were interested in how stem cell research could be used to find out more about the disease and possibly work towards developing therapeutics. I was interviewed and this will be displayed on the support group web site and will be used at support group information days.

None yet.
Year(s) Of Engagement Activity 2009
 
Description Presentation to school children at the Museum of Science and Industry, Manchester (MOSI) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact I was one of two academics presenting 'Stem cell science' to a group of 14-16 year old school children as part of a discussion day organised by MOSI. The presentation was followed by a workshop where the children discussed the ethical implications of working with stem cells and the possible benefits for society.

We received good feedback from the students and the event is now scheduled to run every year.
Year(s) Of Engagement Activity 2008
 
Description Public lecture and discussion - Biochemical Determinants of Tissue Regeneration 
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 Lively dicussion and follow-up visits with local interest groups and schools.

We now have a good interaction with the local popular science interest groups and with the schools that participated in the event.
Year(s) Of Engagement Activity 2013
 
Description School Visit, Bury 
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 I visit this school every other year to talk about my career with A level students. The discussion is wide-ranging including my choice of research area, the role of women in science and career prospects in the current economic climate.

I have been contacted by students from the school and have provided them with information about summer placements etc.
Year(s) Of Engagement Activity 2009,2011
 
Description Stem Cell Debate Day 
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 The University of Manchester host Stem Cell Debate Days for A-Level students from local schools. I participate in these as an expert with experience of stem cells and tissue engineering. Each day hosts 30-50 students and we hold 3 or 4 a year. The students work through case-based scenarios and take part in facilitated debates.

We receive substantial positive feedback from the students on the day of the debate and have also had positive feedback from the teachers.
Year(s) Of Engagement Activity 2009,2010
 
Description Wonder 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Multiple members of the group participated in 'Wonder 2017' the University of Nottingham science spectacular event. We were involved in displays and workshops relevant to our research in regenerative medicine, stem cell biology, animal replacement tools and next generation biomaterials discovery.
Year(s) Of Engagement Activity 2017