The design and analysis of synthetic substrates for embryonic stem cell culture

Embryonic stem (ES) cells have the ability to become any of the thousands of different cell types of the human body. This unique ability means that they have great potential for treating several serious diseases, the current treatments for which are often incomplete, transitory or even non-existent. These include diabetes, Alzheimer's and Parkinson's disease all of which result from cell degeneration or malfunction due to age, infection or injury. However two major obstacles in the development of such ES cell-based treatments are that we still have at best a fragmentary understanding of firstly how to obtain adequate numbers of undifferentiated ES cells and maintain them in tissue culture, and secondly how to control their differentiation in order to produce specific cell types. ES cells are derived from either a single cell or the very small numbers of cells present in the preimplantation embryo, meaning that their numbers have to be amplified in tissue culture in order to produce the amounts that will be necessary for clinical use. This gives rise to the first obstacle because in order to do this, the ES cells currently have to be cultured in the presence of other cell types or animal products. These additions to the culture environment expose the cells to potentially harmful viruses or other infectious agents which could be transferred not only to the patients but also others with whom they come into contact. The second obstacle of not fully understanding how to control the differentiation of ES cells also arises because we know little about the factors supplied by the feeder cells. The aim of this project is therefore to eliminate the need for exposure to other cell types and their undefined products in order to develop a method that will both produce large numbers of uncontaminated ES cells suitable for clinical use and will also allow us to determine the mechanisms that control the differentiation of the ES cells. Work by ourselves and others has shown that one of the crucial factors influencing the behaviour of ES cells in the tissue culture environment is their contact with each other and with the substrate on which the cells grow. We are able to modify the surface properties of materials in controlled ways to produce substrates with specific chemistries and nanotopographies (surface architecture on a scale less than one millionth of a meter), and have shown that not only the chemical composition but also the surface architecture of the substrates does indeed affect cell behaviour. We hypothesise that ES cells can be propagated on synthetic substrates that are designed to control interactions with the cells and thus remove the need for animal products in the culture medium. In the project we will study the surface properties of these materials in detail and evaluate the behaviour of ES cells cultured on them in order to select a set of substrates that show potential to control the behaviour of ES cells. We will then thoroughly investigate the surface chemistry and topography of the substrates and the behaviour of the ES cells exposed to them so that we can design specific surfaces to optimize the ES cell response. This will also allow us to build up an understanding of the mechanism regulating ES cell growth and the mechanisms by which the substrate surface properties control their growth. Such information will facilitate the future production of ES cells suitable for clinical use.

The overall aim of this project is to develop synthetic polymers with controlled surface chemistry and topography both to provide substrates that will facilitate ES cell propagation under GMP conditions (Good Manufacturing Practice) and to permit analysis of the intracellular mechanism that control ES cell self-renewal. The project comprises three complementary activities: substrate synthesis and screening, physical analysis of substrates, and analysis of the cellular consequences of substrate interactions. 1. Substrate synthesis and screening. Following preliminary selection of substrates assayed exclusively with mES cells in order to discard inappropriate (toxic or nonadhesive) candidates, we will screen the effectiveness of designer substrates on both mouse and human ES cell culture systems throughout the project in order that we do not miss species-specific substrates. Screening and substrate development will be an iterative process of increasing structural refinement informed by structural analysis and assays of stem cell pluripotency with increasing stringency. Thus, initial simple assays of ES cell self renewal on candidate substrates and physico-chemical analyses will be used to refine substrate design for subsequent rounds of screening using more stringent criteria, optimized substrates being tested for their ability to support ES cell derivation, long-term culture and maintenance of pluripotentiality. Modifications of substrate chemistry (using simple gas plasmas and proprietary acrylic based coating technology) and topography (using nano particles and nano fibres) will be performed to develop the optimal synthetic substrates that may be applied for subsequent use in the development of GMP conditions for the derivation and maintenance of undifferentiated human ES cells. 2. Physico-chemical analysis of substrates Elucidation of the fine details of the physico-chemical substrate properties that regulate ES cell behaviour is necessary (a) to aid the systematic modifications (tailoring) of substrate candidates to optimize their biological activities and (b) to determine the molecular mechanisms whereby substrates regulate that behaviour. State of the art electron spectroscopy will be used to obtain a fundamental understanding of the local electronic structure and surface functional groups, which in combination with wettability and topography analyses, will determine the requirements for this behaviour. Secondary ion mass spectroscopy will be used for surface chemical analysis of plasma treated surfaces and scanning electron microscopy and atomic force microscopy will be used to obtain high resolution images of the surfaces. 3. Cell biological analyses We will determine the effect of substrate chemistry and topography on the adhesion, cytoarchitecture and cell signalling pathways involved in ES cell self-renewal. In particular the effect of substrates on pathways known to regulate both mES and hES self-renewal will be investigated in order to elucidate the mechanisms fundamental to ES cells in general. Thus, the role of Wnt signalling will be investigated to determine if the main function beta-catenin in self-renewal is to induce transcription of target genes or promote cell-cell adhesion. The activation and localisation of cYes, (also essential for both human and mouse ES cell self-renewal) will be characterised, and we will determine if the requirement for cYes can be obviated if ES cells are grown on permissive substrates. The role of Rho GTPases in mediating these effects will then be investigated, and we will determine if permissive substrates and LIF regulate the same set of Rho GTPases in mouse ES cells. When combined with detailed physico-chemical analysis of the synthetic substrates this will delineate mechanisms by which cell/substrate interactions regulate ES cell self-renewal.