Identifying chemical agents that affect human embryonic stem cells in vitro and determining their molecular targets.

Cells use complicated biochemical mechanisms to control their behaviour and tools that interfere with these processes allow scientists to examine how cells work. This leads to a better understanding of biology and in some cases, can provide new diagnostic and treatment methods for disease. Stem cells, and in particular, embryonic stem cells have been the focus of intense scientific research over recent years. Stem cells are defined by an ability to self renew- a process of division that produces two new cells that are identical to the original. This is important in generating an appropriate number of cells during embryonic development and in adult systems where cells are regularly lost. Stem cells can also differentiate- a process of change that leads to the formation of a cell type whose characteristics are different to the original. This generates the diversity of cells (e.g. muscle, nerve, liver) required to form and maintain the organs and tissues of the body. Another possibility is for stem cells or their derivatives to undergo programmed cell death- a process called apoptosis- which prevents over-production of cells and can eliminate those that have suffered severe damage. Maintaining the correct balance between self renewal, differentiation and apoptosis is an essential feature of multicellular organisms. At the most basic level, this balance is controlled by gene expression. Selective use of some but not all parts of the cells' DNA causes changes in the relative amounts of particular proteins that are produced. This, in turn, affects biochemical reactions within cells and alters their behaviour. A number of methods have been developed that can artificially control gene expression, allowing the function of individual genes to be examined. However, the techniques are time consuming and in some cases, unreliable or their effects short lived. Methods that avoid these problems would help scientists carry out their research more effectively. One possibility is found in the form of chemical agents. Because of their enormous diversity of shape, size and composition, chemical agents are not limited to affecting genes and gene expression. With appropriate molecular structures, any cellular component can theoretically be targeted and its function modified. Also, chemicals can simply be added to a biological system without any requirement for complicated preparation, and the scale of the effect can be controlled by varying the dose. Finding chemicals that are capable of producing effects on specific biological targets is difficult. Methods to design chemicals from scratch that alter the function of particular components of a cell are not reliable. Instead, the most successful approaches involve testing a large number of different, ready-made chemicals to identify those that cause a change in behaviour within a biological system; this may be a whole animal or alternatively, cells grown in isolation in a special container (cultured cells). In order to work as effectively as possible, the testing process must use a biological system that is both sensitive and also relevant to the cells or organism in which the chemicals will eventually be used. With these considerations in mind, human embryonic stem cells are likely to be particularly suitable for finding chemicals that affect the behaviour of human cells. Embryonic stem cells are cultured cells derived from embryos that provide a model of the development process. Like embryos, they are highly sensitive to molecules that affect self renewal, differentiation and apoptosis. The planned project will investigate the behaviour of human embryonic stem cells following treatment with a large number of different chemicals that are likely to interfere with key biological processes. Based upon the results of this work, new ways of both investigating and controlling these and also other human cell types can be developed, benefiting both science and medicine.

The processes of self renewal, differentiation and apoptosis are key areas of interest for stem cell biologists. A number of developmentally important signalling pathways are implicated in determining cell fate with respect to these decisions, including Notch, Wnt, Hedgehog, BMP and TGF?. Conventional methods used to investigate the roles of these pathways include genetic modification, transient gene (over)expression and RNAi. These techniques allow transcription levels to be altered, or the character of a gene product to be qualitatively changed. However, they are subject to significant limitations; for the most part, they are time consuming and in some cases, their effects may be of variable efficiency and short duration. Also, they are not able to directly alter the function of non-protein biomolecules. An alternative method is to use chemical tools; the diversity of chemical structures provides unlimited possibilities for molecular interactions that can perturb the normal activity of biological systems. Also, chemical tools can target any molecular entity within a cell, are easy to apply and are controllable through dose changes. Bottom-up chemical design and synthesis techniques for targeting pre-determined cellular components have met with little success. Currently, the most viable strategy is a top-down screening process in which ready-made chemical libraries are tested for specific effects on in vitro or animal systems. In such a screening process, the sensitivity of the biological system used and its relevance to the intended final recipient system are of the utmost importance. With respect to these considerations, human embryonic stem cells offer an in vitro screening system that is particularly well-suited to the discovery of chemical tools. Human embryonic stem cells undergo multilineage differentiation and also behavioural changes in response to appropriate cues, such as bioactive chemicals. Maintenance of the stem cell state, neural differentiation and apoptosis in the HESC system are each associated with specific cell surface antigens: SSEA3 is found on stem cells and lost upon differentiation, A2B5 is a neural lineage marker and AnnexinV is indicative of programmed cell death. Antibodies recognising these markers can be used to monitor changes in HESC behaviour when experimental conditions are applied. The proposed project will expose HESCs and their derivatives to a large number of candidate bioactive chemical agents and ascertain their effects upon cell behaviour and subsequently, upon specific developmental important signalling systems. The rationale for this approach is that it that chemicals that interfere with critical cellular control mechanisms will produce a phenotype that is detectable by the proposed panel of antibodies. Accordingly, only those candidates highlighted in the first pass (phenotypic) screen will be carried forward for further analysis of developmental signalling pathway activity. The chemical agents to be tested are contained in six libraries; three from a commercial source and three that have been developed internally. In the former category, these are: i) Bioactive lipids; ii) Phosphatase/ kinase inhibitors and iii) Known Bioactive small molecules. In the latter category: iv) diadenosine polyphosphates; v) calmodulin inhibitors and vi) phosphoglycerol kinase ligands. Members of each of these chemical libraries are known to affect self renewal, differentiation and apoptosis and as such, are highly relevant to HESC biology. The proposed project will identify chemical tools that are applicable not only to the investigation of biological systems in general, but which are also especially relevant to the field of HESC research and addressing the problems that currently affect the use of these cells. These include controlling maintenance of the stem cell state and differentiation and also the development of animal free culture methods.