Self assembling peptide hydrogels for human stem cell culture

Lead Research Organisation: University of Manchester
Department Name: Materials

Abstract

The majority of cell culture in vitro takes place on 2D surfaces. This is far removed from the complex 3D environment in which cells naturally reside. An improved understanding of the mechanisms which underlie processes such as cell proliferation, protection from cell death and differentiation of stem cells implicates 3D cell growth as being an important factor in regulating cell behaviour. There is an unmet need in the regenerative medicine, cell and tissue engineering and toxicity screening fields for robust, animal component-free methods for the 3D culture of stem cells. Stem cells have tremendous potential as a source of cells from which cells and tissues needed for transplantation could be derived. This is particularly the case when considering diseases for which there is no current cure (e.g. Parkinson's disease and diabetes). However, there are a number of significant hurdles to be overcome before stem cells can be used for these applications, including the development of methods to allow the large scale expansion of stem cells in culture and protocols for their effective and efficient differentiation to generate cells and tissues of interest.

Hydrogels have emerged as an attractive environment in which to culture cells in 3D. Hydrogels share many qualities with natural tissue and can be used for both the expansion of stem cells and as means of supporting their directed differentiation. There are a number of hydrogels already on the market for in vitro use and some are in clinical trials or already in use in patients for a variety of applications. The peptide hydrogels we will use in this project have specific characteristics which set them apart from these competitors. The peptide monomers used to create the hydrogel are simple, short and relatively cheap to manufacture. The simple eight amino acid building blocks (constructed of alternating hydrophobic and hydrophilic residues) are fully synthetic and free of animal products and they can easily be modified to contain bioactive motifs which influence cell behaviour. We have therefore developed a protocol which allows the use of these simple peptide hydrogels for the culture of mouse embryonic stem cells (mESCs). The mESCs had excellent viability within the gels and retained their stem cell characteristics. Cells can be serially passaged in the gels, recovering intact cells from one gel before re-seeding them into a new gel. Importantly, the protocol we have developed ensures that the gels are reproducible and transferable to any lab with a standard skill set. Preliminary experiments suggest that the gels can also be used for the 3D culture of human ES cells (hESCs) thereby transforming a research tool to a possible answer to the unmet need detailed above. The mechanical properties of the gels can be altered to make them similar to the various tissue environments found within the body and the gels have the additional quality of being injectable. These are characteristics which extend their potential application, but are not something we will address in the current project.

The project will focus on providing the proof-of-concept data we need to make our technology attractive to industrial partners to enable us to move towards commercial exploitation of our research. In addition, we will work closely with clinicians and other end-users of the technology to ensure that we are developing gels with the required characteristics for a wide variety of applications. On completion of the project we will be in a strong position to secure support from translational funders (for example MRC DPFS, NIHR i4i), venture capital or seed funds and will have an in-depth knowledge of the possible market opportunities and regulatory landscape for our products.

Planned Impact

While the use of stem cells in regenerative medicine and toxicology has great potential, critical issues must be addressed to both move cellular products into the clinic and to effectively screen new drugs/toxins. Particularly important is the capacity to produce robust, reproducible protocols for the good manufacturing practice (GMP) compliant production and maintenance in vitro of seed stock cells and their differentiated/manipulated progeny. This project will use peptide hydrogels for the tissue-realistic 3D culture of hSCs. The hydrogels share similarities with the native cell environment but are fully-defined, synthetic and animal product-free. The outcome will be a platform technology which can be licensed for the development of stem cell therapies and drug testing that advance the field of human stem cell culture and the UK position at the forefront of this field. We already have a major chemical company as a partner to use the gels for in vitro differentiation of mES cells to chondrocytes. However, the work outlined in this project will enable us to understand all potential applications of our technology and to identify and start to work with a variety of end-users (clinicians and industrial representatives) to ensure that we develop gels with broad appeal to many market sectors. These can be segregated into those which would be available in the short, medium and long term - primarily based on the regulatory restriction placed on various applications. Examples of some of these are discussed below and these are shown diagrammatically as part of the TTO letter of support.
In vitro research applications and in vitro stem cell expansion (short term - at the end of this project): The cell culture market is dominated by a few large companies. There are also key players who target the stem cell market specifically. Current expenditure on 3D cell culture environments for stem cells is hard to estimate but most of the major companies now have products in this area. We anticipate that our gels with their reproducibility and stability will make a significant impact in this area.
The major market opportunity is in cell/tissue regeneration (medium term - following 2-3 years development after this project). The global market potential for stem cell therapies is predicted to increase substantially given the large patient numbers amenable to cellular therapies. A recent report (Business Insights, 2009) evaluated the 2008 worldwide market at US$410m and forecast it to be worth US$5.1bn by 2014. At present, the USA dominates the market (90%), with the UK as the 4th world market leader in stem cell therapies. Our gels can be seen as a 'blank canvas' to which lineage-specific cell-binding or cell-signalling motifs could be attached. This project aims to link our technology to key end-users who will be integral in supporting the development of the gels for the production of various cell/tissue types. Our goal is to seek additional funding to establish a core research facility to optimise the gels prior to licensing modified gels to end users.
There is considerable interest in the use of stem cells as drug discovery and testing tools (medium term), driven by the observation that ~80% of compounds fail during clinical development as a result of unexpected toxicity. There is a need for 3D culture environments for the tissue-realistic growth of cells in a format amenable to automation. Our gels are suitable for this application. There would need to be an additional period of technological development to allow the design of high-throughput cell assays in the gel-based 3D format and we would seek additional translational funding to support this.
Direct in vivo application of the gels, for example used to support the localisation of cells to a target site as an injectable scaffold or used as an implanted scaffold for engineered tissue are future applications which, while important to be considered during development are long term products.

Publications

10 25 50
 
Description The hydrogels are able to support the culture of human cells in a 3-dimensional format, which is similar to the natural environment of cells within the body, unlike traditional culture systems which are mostly 2D. The hydrogels are highly reproducible which makes them useful for experiments were we need to be sure our results are accurate and where we are using expensive or hard-to-obtain samples. In addition, the hydrogels are useful for a wide range of applications across the fields of tissue engineering and regenerative medicine as well as basic cell biology/biochemistry.
Exploitation Route We are actively collaborating with other accademic, clinical and commercial partners to continue to develop these hydrogels for a wide range of applications. We have also continued to protect the intellectual property relating to the cell culture application of these hydrogels and a patent application has been submitted in North America and in Europe.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description During the funding period of this grant we were able to optimise the use of the hydrogels for culture of human embryonic and adult stem cells. We were also able to develop methods for imaging cells growing within the gels and for their extraction for further study. The work undertaken as part of this follow-on-fund led directly to futher funded work, including an award from the University of Manchester IP company (UMIP) to investigate directed differentiation of human embryonic stem cells to generate hepatic cells suitable for drug screening applications. In addition, a number of collaborations were initiated with other groups and companies to maximise the use of the hydrogels across multiple applications.
First Year Of Impact 2013
Sector Chemicals,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Economic

 
Description Application of a 3D hydrogel-based model to replace use of animals for passaging patient-derived xenografts
Amount £90,000 (GBP)
Funding ID NC/P002285/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2020
 
Description Biochemical Society Workshop
Amount £30,000 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2017 
End 05/2017
 
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 Public
Country United Kingdom
Start 03/2017 
End 03/2020
 
Description Experiencing the micro-world - a cell's perspective
Amount £1,300,000 (GBP)
Funding ID EP/R035563/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 11/2021
 
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 MCR studentship
Amount £72,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 08/2017 
End 08/2020
 
Description NC3Rs PhD Studentship
Amount £90,000 (GBP)
Funding ID NC/P002285/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Public
Country United Kingdom
Start 03/2017 
End 03/2020
 
Description NC3Rs Project
Amount £334,000 (GBP)
Funding ID NC/N001583/1 
Organisation National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) 
Sector Public
Country United Kingdom
Start 02/2016 
End 02/2019
 
Description Stoneygate Childrens' Brain Tumour Research Fund
Amount £622,533 (GBP)
Organisation Stoneygate Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2016 
End 08/2020
 
Description The Leverhulme Trust International Travel Fellowship
Amount £30,000 (GBP)
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2014 
End 06/2015
 
Description The Leverhulme Trust Visiting Professorship
Amount £18,720 (GBP)
Funding ID VP-2016-014 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2017 
End 07/2017
 
Description UMIP Proof of Principle Fund
Amount £101,000 (GBP)
Organisation University of Manchester 
Department Innovation Group (UMI3)
Sector Private
Country United Kingdom
Start 01/2013 
End 01/2014
 
Description University of Nottingham Regenerative Medicine RPA
Amount £10,000 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 08/2017 
End 03/2018
 
Description University of Nottingham Regenerative Medicine RPA
Amount £24,369 (GBP)
Organisation University of Nottingham 
Sector Academic/University
Country United Kingdom
Start 03/2017 
End 01/2018
 
Title Peptide gels for 3D breast cancer models 
Description Current materials used for in vitro disease models are often limited by their poor similarity to human tissue, batch-to-batch variability and high complexity in composition and manufacture. We have developed a "blank slate" culture environment that can be customized by incorporating matrix components specifically selected to match the target tissue, with mechanical properties controlled independently and simultaneously. Based on a self-assembling peptide hydrogel, this system contains no exogenous proteins or glycosaminoglycans: only those specifically added, or those synthesized by the cells in culture. This 3D culture platform therefore provides full control over biochemical and physical properties, allowing the composition and mechanics of the tissue of interest to be recapitulated in vitro. As proof-of-concept, we designed a panel of hydrogels designed to mimic the stages of breast cancer progression. Controlling the peptide gelator concentration allows hydrogel stiffness to be matched to normal breast (<1 kPa) or breast tumour (>1 kPa), with higher stiffness favouring the viability of breast cancer cells over normal breast cells. In parallel, these hydrogels may be modified with matrix components relevant to human breast, such as collagen I and hyaluronan. The choice and concentration of these additions control the size, shape and organisation of the breast epithelial cell structures formed in co-culture with fibroblasts. This system therefore provides a means of unravelling the individual influences of matrix, mechanical properties and cell-cell interactions in cancer and disease. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2018 
Provided To Others? Yes  
Impact We have presented the gel technology at events specifically designed to encourage uptake by other researchers working in this area (e.g. BACR meeting in Leeds, May 2018) and have trained researchers from other groups to encourage uptake of the technology. These training visits were supported financially by NC3Rs. We have also developed additional collaborations related to this technology including with a commercial partner able to provide the raw materials required for the gels. By demonstrating the applicability of the peptide gels to breast cancer modelling we have also attracted additional collaborations with groups working on fibrosis and the support of stem cell differentiation. 
 
Description Coyle 
Organisation University of Nottingham
Department Division of Child Health, Obstetrics and Gynaecology
Country United Kingdom 
Sector Hospitals 
PI Contribution In this collaboration I provide matrix biology expertise, stem cell biology expertise and materials science expertise to generate in vitro models of childrens' brain tumour.
Collaborator Contribution Dr Beth Coyle is a long-standing expert in childrens' brain tumour, investigating the mechanistic basis of initiation and spread of disease.
Impact The collaboration is multi-disciplinary linking cell biology, cancer biology and materials science. Together we are developing novel models of childrens' brain tumour.
Start Year 2017
 
Description Farnie 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution I provide hydrogel and extracellular matrix expertise to help generate 3D in vitro models of breast cancer.
Collaborator Contribution The breast cancer group provide expertise in the study and characterisation of breast cancer as well as access to patient material which we then use to create in vitro models of breast cancer.
Impact We have been awarded an NC3Rs project grant to develop hydrogels as replacements for in vivo models of breast cancer (started Feb 2016). This collaboration combines biomaterials science, matrix biology and cancer biology.
Start Year 2014
 
Description Neil Thomas 
Organisation University of Nottingham
Department School of Chemistry Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have started working with a group based in Chemistry at Nottingham. The group have expertise in chemical functionalisation strategies that we can apply to our peptide gels. We are able to provide a biologically relevant test system in which to demonstrate the utility of their methods.
Collaborator Contribution The chemists have already developed a variety of methods for functionalization of peptide fibres and this technology can be transferred to our peptide gels.
Impact This collaboration links organic chemistry (Neil Thomas) and matrix biology (Cathy Merry). We have recently been awarded a BBSRC iCASE studentship with which to follow up on our preliminary work.
Start Year 2018
 
Description Pepceuticals 
Organisation Pepceuticals
Country United Kingdom 
Sector Private 
PI Contribution We are working with this company, using the peptides they synthesise to create 3D hydrogels for cell encapsulation.
Collaborator Contribution Pepceuticals are a synthetic peptide company and are interested in novel products.
Impact We have recently been awarded a BBSRC iCASE studentship (PI is Neil Thomas, Cathy Merry CoI with Kamal Badiani from Pepceuticals) to create novel functionalised peptides that can be applied to the peptide gel system. This links organic chemistry (Neil Thomas), matrix biology (Cathy Merry) and peptide synthesis at a commercial scale (Kamal Badiani).
Start Year 2018
 
Description SPG 
Organisation Uppsala University
Country Sweden 
Sector Academic/University 
PI Contribution I am hosting a visiting fellow supported by an award from the Swedish Research Council (Vetenskapsradet)
Collaborator Contribution The fellow will work to develop iPSC models of heparan sulphate-related disease using the hydrogels.
Impact None
Start Year 2016
 
Description Whitelock 
Organisation The Leverhulme Trust
Country United Kingdom 
Sector Charity/Non Profit 
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 METHOD OF MAKING A HYDROGEL 
Description The present invention relates to a novel protocol for making a hydrogel, which shows increased stability compared to hydrogels of the art, and can be reliably reproduced. The hydrogels produced by the methods of the present invention are preferably three dimensional, and particularly suitable for the culture of stem cells. 
IP Reference WO2013124620 
Protection Patent application published
Year Protection Granted 2013
Licensed No
Impact Further funding to develop specific applications of the technology
 
Title NEW SCAFFOLD FOR CARDIAC PATCH 
Description A biocompatible and biodegradable medical device patch actuating primarily as soft tissue structural reinforcement. The device has a layered architecture, where the primary serves as suturing layer and mechanical support to a thick porous scaffold which can be coated with a mimic-like extra cellular matrix (ECM). The device can be provided to the end user under the format of independent layers that can be cut and assembled to the specific need to the end user and patient. The layers are assembled without the need of any adhesive. Totally haemocompatible and of behavior superior to polytetrafluoroethylene used for any soft tissue repaired, the field of this invention is demonstrated for cardiovascular therapy but should not be limited to it. It is of practical relevance of vein, tendon and hernias and dermal treatments. 
IP Reference WO2014044321 
Protection Patent application published
Year Protection Granted 2014
Licensed No
Impact None so far.
 
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 NC3Rs/Unilever joint workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This was a scoping workshop to identify opportunities for NC3Rs to work towards the development of animal product free in vitro systems. This involved a variety of industrial and academic partners looking at various 'needs' where we could/should be developing alternatives to animal use. My participation was focused on non-animal alternatives to Matrigel.
Year(s) Of Engagement Activity 2019
 
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 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
 
Description Wonder 2019 
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 Public/other audiences
Results and Impact We ran a stall at the University of Nottingham's premier outreach event 'Wonder'. We used the stall to highlight two aspects of the group's work. One was the use of peptide hydrogels to create improved models of disease (primarily cancer). The other was the importance of sulphated glycosaminoglycans in health and disease. Both aspects were very popular - we had hands-on exhibits targetted at children and additional information available for older children/adults so we had a lot of engagement and positive feedback. Mostly this was raising the awareness of the importance of the 3Rs in laboratory work and cancer research in particular.
Year(s) Of Engagement Activity 2019
URL https://www.nottingham.ac.uk/wonder/