Biocompatible Sterilisable Worm Gels: An Enabling Technology for the Development of Pluripotent Human Stem Cell-based Therapies

Human embryonic stem (hES) cells are pluripotent cells that can either self-renew, thereby maintaining their pluripotency, or differentiate depending on the culture conditions. Induced pluripotent stem (iPS) cells, which offer similar clinical potential to hES cells, can be generated by infecting adult cells. In principle, the application of hES and iPS cells in cell therapy and regenerative medicine offers tremendous potential because of their innate ability to differentiate into multiple, clinically-useful cell types. However, well-defined culture conditions are essential for realising the biomedical potential of hES and iPS cells. Matrigel is a gelatinous protein mixture secreted by mouse sarcoma cells and is marketed by BD Biosciences. This complex mixture contains laminin, entactin, collagen and various growth factors; it resembles the complex extracellular environment found in many tissues and is used by cell biologists as a model active substrate for cell culture studies. Matrigel is a liquid at 4oC, but on warming to 37oC it forms a fibrillar gel network. In its diluted form, Matrigel is used as an attachment substrate for culturing embryonic stem cells to maintain their pluripotent, undifferentiated state in the absence of any feeder cells. Despite its high cost, animal origin and poor/variable batch-to-batch reproducibility, Matrigel is nevertheless widely used by cell biologists. However, an alternative wholly synthetic gelling composition that can be reliably employed as a baseline material is urgently required for a wide range of in vitro stem cell experiments aimed at eventual clinical applications. Moreover, it is widely accepted that the effective translation of human pluripotent stem cells into cell therapies will require the development of standardised tests for product consistency, stability, toxicity and immunogenicity.

With the aid of this grant, we will develop a range of novel, wholly synthetic hydrogels based on the self-assembly of biocompatible methacrylic block copolymer worm-like particles, which are readily prepared in concentrated aqueous solution. Such gels are highly biocompatible and, unlike many hydrogels, can be readily sterilised simply by cold ultrafiltration: this is possible because the worms transform into free-flowing spherical nanoparticles when cooled to 5oC and reform worm gels on returning to ambient temperature. In a year-long informal collaboration, we have conducted proof-of-concept studies (see A. Blanazs et al., JACS, 2012, 134, 9741) and filed a U. Sheffield patent application, thus we already have a strong background IP position. However, there are many remaining technical challenges and a concerted inter-disciplinary research effort is now required to overcome these problems. Our new worm gels are expected to replace Matrigel (and related animal-derived materials) as the most convenient medium for the long-term storage, manipulation and proliferation of human stem cells while retaining their pluripotent state. Prof. Steve Armes will lead on the synthetic polymer chemistry aspects of this inter-disciplinary study, while Prof. Harry Moore will lead on the stem cell research. We request funding to support two experienced post-doctoral research scientists to work in close collaboration on this project. We have identified two appropriate industrial partners for this EPSRC grant. GEO is a UK-based speciality chemicals company that will provide the monomer building blocks required for the synthesis of the block copolymer worms, assist with the scale-up studies and act as a raw materials supplier in the event of future commercialisation. Plasticell is a UK-based biotech SME specialising in stem cell technologies. This company is ideally placed to help us assess and optimise our worm gels to ensure that they provide an appropriate technical solution for stem cell biologists. These two companies have each pledged £ 5 K cash to provide the £ 10 K contribution required by EPSRC.

Prof. Steve Armes has worked extensively with many industrial companies. Current sponsors include DSM, P & G, BP, Lubrizol, Ashland, Scott Bader and GEO. He sold a U. Sheffield patent application to DSM for 125,000 euros in Sept 2007. This technology is now the basis of a successful anti-reflective coatings business for DSM (tradename - Khepricoat; see www.dsm.com), who describe its interaction with Prof. Armes as an exemplar of its 'open innovation' policy. As a named inventor on 20 patent applications, Prof. Armes is well aware of the value of IP protection and is undoubtedly 'outward-facing' regarding the potential commercial impact of his work. He has recently filed three U. Sheffield patent applications arising from his EPSRC-funded research programme on polymerisation-induced self-assembly, which underpins the synthesis of the block copolymer worm gels described in the present grant application. One of these patent applications describes the survival of human stem cells in worm gels over a three-week period without differentiation and was filed jointly in March 2013 with Prof. Harry Moore. Thus U. Sheffield has already secured a useful independent background IP position on the basis of our proof-of-concept experiments. Prof. Moore has worked with a range of bioscience companies, including a recent consultancy with Pfizer. This company subsequently scaled up the GMP-compliant manufacture of Prof. Moore's Shef1 cell line and now plan to treat age-related macular degeneration (blindness) in a human clinical trial scheduled to start in 2013. Thus Prof. Moore is also well used to fruitful collaboration with companies. In summary, both lead academics have the appropriate technical backgrounds and commercial experience to ensure that the translational aspects of this grant proposal bear fruit.

GEO has worked with Prof. Armes for 17 years. This speciality chemicals company is currently part-funding a PhD student (Joe Lovett) in the Armes group, who is working on pH-responsive worm gels. GEO manufactures both glycerol monomethacrylate and 2-hydroxypropyl methacrylate and is willing to provide these two monomers for the synthesis of the block copolymer worm gels. This company also has substantial experience in efficiently transferring complex chemistry from the laboratory to multi-ton manufacturing scale while maintaining GMP standards and would be a suitable raw materials supplier in the advent of commercialisation.

Plasticell is a pioneering UK-based biotech SME which specialises in the differentiation of stem cells to obtain cell lines for sale to pharma companies. It uses its proprietary bead-based combinatorial screening platform (CombiCult) to differentiate induced pluripotent stem cells, embryonic and adult stem cells into various somatic lineages. Plasticell is also developing its own alginate-based gel for stem cell culture, so it is well placed to assess the commercial potential of our worm gels. Plasticell has used Prof. Moore's Shef cell lines for various in-house experiments for a number of years and this company is also an industrial member of the Pluripotent Stem Cell Platform (PSCP), a £7.5M Regenerative Medicine Hub led by U. Sheffield and funded by MRC, BBSRC and EPSRC.

Once a strong IP position has been secured, we will publish our results in high-quality journals such as JACS, Angewandte Chem, Adv Mater, Nature Biotech, Cell, Biomaterials etc. We will disseminate our findings at international conferences via talks given by both two post-doctoral scientists and also by the two Principal Investigators.

In 2005 U. Sheffield sold its entire IP rights to Fusion IP in a ten-year pipeline deal worth £10 M. This AIM-listed company has first refusal to commercialise inventions by University employees arising from Research Council contracts. If a spin-out company is not considered the best business option, Fusion IP can provide business expertise to aid the negotiation of patent licences or patent sales.