Self assembling peptide hydrogels for human stem cell culture

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.

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.