Use of bioactive polymers to regulate differentiation of embryonic stem cells in three-dimensional bioreactors

Human embryonic stem (hES) cells, isolated from very early stage embryos, are able to become any cell in the body and have great potential for the treatment of many serious diseases. However there is a practical problem in producing a sufficient number of hES cells to treat human diseases, such as heart disease and cancer. At present hES cells must be grown under specialised conditions as a 2-dimensional (2-D) culture. This severely limits the number of cells that can be obtained and our project will allow the culture of hES cells in 3-dimensions (3-D). To allow you to appreciate the significance of 2-D and 3-D growth of hES cells, imagine how many more tennis balls you could squeeze into your living room compared to those you could only place on the floor. The bigger the volume of the room the more tennis balls you can fit in, irrespective of the area of the floor. Therefore, 3-D growth of hES cells allows us to grow a lot more cells than we can at the present time. However, it's not that easy! If we grow hES cells in 3-D they will either die or turn into other cell types (called differentiation) therefore we need to stop them from differentiating. We will use two methods to stop the hES cells differentiating in 3-D culture. First we will use sugars. These are different to the sugar you put in your coffee since they consist of a long chain containing areas that can regulate biological activities. All the cells in your body naturally contain these sugars and they are very important in maintaining healthy and viable cells. Basically, we will use these sugars to help keep the hES cells feeling well in 3-D culture. The other method we will develop uses oligopeptides. All proteins in your body are made up of amino acids and an oligopeptide is simply a chain of amino acids. Like sugars, peptides can regulate what happens in your body. We will use oligopeptides that make hES cells healthy and allow them to grow in 3-D culture. Sugars and oligopeptides can work well together and we hope that by combining them together we can make the hES cells grow very well in 3-D. In this way we will engineer bioreactors of cells in 3-dimensions that can produce large numbers of hES cells. This will allow us to provide a new technology for mass production of hES cells and then turn them into specific cell types that may help to treat patients with serious diseases.

This project will develop a suspension bioreactor for human embryonic stem (hES) cells, enabling production of large numbers of undifferentiated cells that can subsequently undergo differentiation into all three germ layers (mesoderm, ectoderm & endoderm). We will achieve this using a library of soluble polymers bearing bioactive agents. The use of polymer conjugates will endow the bioactive agents with greater cell-binding activity through increased avidity, and this may be further increased by using polymers with slight positive charge or membrane-inserting pendent groups. We anticipate that this will overcome some of the greatest technical limitations currently associated with hES culture, that of substrate-dependence and the critical importance of cell density. Two types of bioactive agents will be studied. The first are saccharides containing variable sulphation patterns, since there is evidence that 6-O-sulphation within heparan sulphate can modulate the activity of ligands critical for hES biology. For example, loss of 6-O sulfation inhibits FGF-2 activity (important for maintainance of hES cell pluripotency) yet promotes BMP activity (required for mesoderm differentiation) by loss of cell-surface recruitment of BMP-agonists. Once seleced using a small-scale hES monolayer culture system, these oligsaccharides will be conjugated to synthetic polymers, mimicking their display on HSPGs at the cell surface. Their effect on high-capacity non-adherent hES culture will then be evaluated in the bioreactor system. Secondly we will use peptide phage display libraries to identify oligopeptides capable of enabling survival or hES cells in suspension culture (in the presence of optimal oligosaccharide-polymer conjugates) and potentially also inhibiting differentiation. Sequential rounds of biopanning, using fluorescence-linked reporters and fluorescence activated cell sorting to reclaim viable, undifferentiated cells after prolonged suspension culture, will provide enriched phage libraries and we will seek to identify consensus oligopetide motifs. Biological activities of phage clones expressing representative oligopeptides will be confirmed (including assessment of mixtures of clones) and priority oligopeptide sequences will be covalently linked to soluble carrier polymers based on poly[N-(2-hydroxypropyl)methacrylamide] (polyHPMA) using a range of spacer groups. The ability of these polymer conjugates to enable large scale production of undifferentiated hES cells will be assessed. The properties of the carrier polymer may also be manipulated to maximise effectiveness of the polymer conjugates. This will include incorporation of positive charges or pendent membrane-inserting hydrophobic groups. This approach should decrease the amount of polymer conjugate required, and increase the responsiveness of the system when differentiation is eventually induced. Ultimately we will produce a fully synthetic cost-effective bioreactor system for the maintenance of pluripotency during expansion of hES cells.