Development of dynamic 3D culture systems for maintenance and expansion of pluripotent embryonic stem cells

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Stem cells are present within our bodies throughout our lives and are very important because they make sure that as damaged and worn out cells die, there is a supply of new cells to replace them. Some organs and tissues have a tremendous capacity to replace cells, for example the blood system, the liver and the inner lining of the gut. However, other tissues, for example the brain and heart, have almost no capacity for regeneration. Stem cells have been isolated from embryos at very early stages of development (termed embryonic stem cells) and also from a variety of adult sources including bone marrow, gut and muscle. Research has shown that these stem cells have two remarkable properties. They can divide and form two identical stem cells, i.e. self-renew themselves, or they can form many other types of cells by a process called differentiation. There is currently great interest in harnessing these unique properties of stem cells because by using stem cells it may be possible to generate cells outside the body i.e. in the laboratory, that can then be used to replace damaged tissues inside the body. Many chronic diseases cannot currently be effectively treated because loss of cells is the underlying cause. Several brain disorders occur because certain nerve cells die, e.g. in Parkinson's disease. In childhood diabetes, the cells of the pancreas that normally act to control the level of sugar in the blood are destroyed meaning patients are reliant on regular injections of the hormone insulin in an attempt to regulate their blood sugar levels. Stem cell-based therapies offer potentially exciting alternative treatments for the sufferers of such diseases. However, much more research needs to be carried out on stem cells and their behaviour before such advances will be brought into modern day medical practice. The environment that normally supports the growth and survival of stem cells within the body has 3-dimensional architecture and there is increasing evidence that this 3D microenvironment is critical for maintenance of the stem cell phenotype. However, the methods currently used in laboratories to expand and then study stem cells rely largely on 2-dimensional cultures. In this proposal we want to develop ways in which embryonic stem cells can be maintained and expanded in 3-dimensional dynamic culture systems, since this would more closely mimic their natural environment within the developing blastocyst of the host. Such 3-dimensional culture could afford significant advantages over culture in 2-dimensions. We will investigate the ability of scaffolds made of different materials, with different surface modifications, to maintain ES cell growth and self-renewal. We will further investigate the effects of different bioreactor formats and the influence of different growth factors on ES cell maintenance and growth. Our aim is to begin by studying the behaviour of ES cells derived from mice, but in parallel to initiate experiments with human ES cells. We hope that these studies will enable us to optimise the most appropriate dynamic 3D culture system that allows for expansion and maintenance of hES cells. This is an important goal that needs to be addressed if basic stem cell research is to be successfully translated into the clinic. Such expansion is critical for future applications in situations where large numbers of undifferentiated human ES cells are required, for example application of these developments could make it possible to grow large numbers of undifferentiated stem cells that can subsequently be differentiated into e.g. nerve cells, liver cells etc and used to replace damaged cells and tissues in patients suffering from chronic diseases. Such cell-based strategies offer real hope for the sufferers of many such diseases and the research proposed here could be of real benefit in the medium to longer-term.