Prospective isolation of intermediate states during lineage commitment

The different cell types that make up the body all derive from a small number of unspecialised cells that are present at an early stage in embryonic development. Proper development of the embryo depends on these cells becoming incrementally more specialised, in such a way that the right cell types appear at the right place and the right time, yet we do not fully understand how this happens. We aim to identify the factors that drive specialisation (differentiation) of cells in early development. Embryonic stem cells (ES cells) are cells that are taken from the very early embryo and grown in a culture dish. They are capable of making any of the different specialised cell types in the body. We will use ES cells as a model system for identifying the factors that drive early embryonic decisions. Any important findings will then be tested in mouse embryos to see if they hold true during normal development. This work will help us to better understand how different tissues form. ES cells are also useful because they provide a source of specialised cell types that can be used for modeling disease and for testing the effects of drugs. However, attempts to generate useful cell types from ES cells are hampered by the fact that differentiating cultures are generally contaminated with undesirable cell types. This seems to be becuase individual cells within a culture can each respond differently to external signals, and it is currently not possible to predict exactly how any given cell will respond to a particular signal. We hope to discover a predictive marker that we can use to isolate a subpopulation of cells that will respond uniformly and predictably to particular signals. We will use the findings of our work to devise strategies to preditably direct differentiation in all cells within a culture. Our findings, and the tools we will make, will be useful for other scientists in experiments to manipulate and monitor embryonic stem cells and the specialised cells that derive from them. Our tools to monitor ES cell decisions will also be useful for biotechnology companies in their search for compounds that influence the way that cells behave in normal or damaged tissues. This work may teach us how to generate useful cell types from ES cells in a dish, which we could transplant to repair damaged tissues. Also, new drugs could be tested on ES-derived cells in culture before being tested in animals or humans.

Embryonic stem cells are a useful experimental model for studying the mechanisms underlying lineage choice, and are a valuable source of specialised cell types for drug screening and disease modelling. This all depends on our ability to predictably drive and monitor differentiation into desired lineages. However, at present, even under fully defined optimised conditions, we cannot uniformly drive differentiation of a population of ES cells into a single lineage: cultures are inevitably contaminated with undesired cell types. We cannot predict which individual cells will fail to respond appropriately to a given differentiation condition. This underscores the fact that we know relatively little about the mechanisms that direct lineage choice. We have identified three candidate transcription factors that are normally associated with particular differentiated lineages and are present in an inactive state in ES cells. We propose that these transcription factors influence lineage choice, and that variability in their expression between individual cells may help explain variability in the differentiation response. We will test this in two ways: by forcing or suppressing activation of the candidate factors to find out which differentiated lineage they can favour, and by making sensitive fluorescent reporters to test whether these are prospective markers of cells that are biased or committed to particular lineages. We will exploit these factors as markers of early primed or committed states. Our fluorescent reporters will make it possible to monitor these states within mixed cultures in experiments to test extrinsic factors that control differentiation. This would give a much more accurate readout than analysis of a pooled heterogeneous population. We will also be able to isolate these populations and profile their gene expression in order to gain clues as to the intrinsic factors that define these states and the signals that regulate transitions between them.

Who will benefit from this research, and how will they benefit? 1) Academics in stem cell research and developmental biology: The knowledge and tools we will produce will be of immediate benefit to researchers interested in early-lineage specification in ES cells or in vivo, or in neuronal and pancreatic beta cell differentiation, EMT, and tumorogenesis. The way they will benefit is described in detail in the 'Academic Beneficiaries' section. 2) Groups working in technology development: Edinburgh University is strong in developing new technologies for high-throughput discovery platforms (Manfred Auer, Mark Bradley), and we will be in an excellent position to feed our reporter lines directly into these platforms. This provides an exciting opportunity to increase the impact of our research beyond the proposed work within our own lab. 3) Biotechnology industry: Biotech companies will benefit from our reporter lines as cell-based readouts in high-throughput-screens to identify small molecules that can be used to manipulate ES cell differentiation, pancreatic beta cell specification, neurogenesis, and tumour metastasis. The biotech sector will also benefit from our discoveries of additional markers of lineage commitment, including cell-surface proteins that can be used to monitor lineage choice in any line of mouse or human ES cells. Furthermore, our work will provide important clues for the rational design of strategies to control differentiation and EMT. These strategies could be exploited directly for rational drug design, or indirectly through generation of useful ES-derived cell types that will then feed into disease-modelling and drug-testing programmes within the biotech sector. 4) Patient groups: The commercial and academic activities described above will lead towards development of new treatments for human diseases including cancer and diabetes, and may also help deliver ES-derived or iPS-derived cells for regenerative therapies for neurodegenerative disease and diabetes. 5) The wider public: There is considerable interest in stem cell research amongst the public, and this has helped to awaken a wider interest in science in recent years. I have a strong track record in outreach activities and am supported by an impressive communications and outreach programme in our institute. Our research will contribute to ongoing activities that include presentations at science festivals and science centres, adult education courses, web-based education tools, and public workshops and debates. What will be done to ensure that they benefit from this research? The MRC Centre for Regenerative Medicine is well placed to ensure that all of these groups receive maximum benefit from our research. Our centre has strong links with industry and with translational research groups, and specific links for the development of my own research in these areas will be coordinated by the Centre management team. Commercialisation and IP will be managed by Edinburgh Research and Innovation, the commercialisation branch of Edinburgh University. Communications and outreach with public, press, patient groups, school groups, and other target audiences is coordinated centrally by our dedicated communications officer. These activities also benefit from our strong links with international outreach networks arising from the EuroStemCell, ESTools, and EuroSyStem projects.