Systematic Identification of Tunable Transfection Reagents for Stem Cell Biology

Human embryonic stem (hES) cells are of great research interest, and offer the promise of therapeutic value, due to their pluripotency, or ability to develop into one of a large number of differentiated cell types. However, research on ES cells is currently hampered by the low transformation efficiencies observed by standard technologies based on non-viral lipid-mediated DNA delivery. Transformation is the ability to introduce DNA into cells, and permits the over-expression, 'knock-down' or biochemical 'tagging' of a specific protein, and is a standard means to assess the contribution of a given protein to the cell. We propose to overcome this technical barrier by establishing a systematic and results-informed search for new and effective reagents to chemically assist the transfection of stem cells. This will be performed by combining the expertise of two research groups, Cambridge (Florian Hollfelder) has broad experience of chemical synthesis, library development and high throughput screening, whereas Birmingham ( Karl Nightingale & Laura O'Neill) have research programmes centred on the use of primary and established leukaemic cell lines together with mouse and human ES cells. Specifically, we propose to generate a broad and chemically diverse library of modified polyethylene imines (PEIs), a reagent widely used in cell transfection, and establish a high throughput screen to identify effective and non-toxic reagents. Effective reagents will be subjected to structure/activity analysis to inform secondary library synthesis if necessary. This library will be screened against a number of tissue culture cell types of increasing technical difficulty. Initially we propose to use a leukaemic cell line called HL60 cells. This represents an ideal cell line for troubleshooting the screen as it grows quickly and easily, but is also a difficult cell type to transfect - this should be useful in establishing screen conditions for the stem cells. Importantly, any reagents identified will of immediate general interest as HL60 cells are widely used in research labs, being a good model of acute myeloid leukaemia. We then propose to utilise the experience gained from this initial phase to re-screen the library against several sorts of mouse ES cells, and finally against several sorts of human ES cells. An important part of the proposal at that the identified reagents will be subjected to a number of 'quality control' checks. This will primarily be at a functional level (or ensuring that the cells continue acting like stem cells), but we will also check that the reagents can perform the sorts of experiments that molecular biologists would want to use them for.

Human embryonic stem (hES) cells represent a significant research tool for dissecting developmental pathways, and offer great promise for therapeutic intervention. However, research on ES cells is currently hampered by the low transformation efficiencies observed by standard ('lipofectamine')-assisted technologies. Advances in this field are clearly required as this is an identified 'Scientific priority' for this initiative. We propose to overcome this technical barrier by generating a diverse library of polyethylene imines (PEIs), and Gemini surfactants, both classes of reagents with demonstrated transfection function. These libraries will be subjected to a systematic, reiterative screen by an established high throughput procedure based on GFP expression. Structure-activity relationship analysis will inform secondary library synthesis if required. We propose to use this procedure for screening a number of cell types of presumed increasing technical difficulty. Initially we propose to use a leukaemic cell line (HL60's). This represents an ideal 'troubleshooting' line since it grows in suspension, divides rapidly, and yet is noted for poor transformation efficiencies with current chemical reagents (lipofectamine). Importantly, any reagents identified will of immediate general interest as the line is widely used, being a good model of acute myeloid leukaemia. We then propose to utilise the experience gained from this initial phase to re-screen the library against a number of male and female mouse ES cell lines, and finally against several human ES cell lines. An important part of the proposal is that .identified reagents will be subjected to a number of 'quality control' checks. This will primarily be at a functional level, by ensuring that 'mock-transfected' cells (ie transfected without DNA) remain undifferentiated, but will progress down defined differentiation pathways given the appropriate cues.