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

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.

The ability to maintain stem cell pluripotency and direct lineage specific differentiation are key goals in current stem cell-based research. It is clear that the stem cell microenvironmental niche plays a key role in determining stem cell behaviour. Such stem cell niches are difficult to mimic in the 2D cultures widely used to propagate undifferentiated ES cells. The use of extracellular matrix-like 3D scaffolds could, therefore, provide a powerful alternative approach to mimic the stem cell microenvironment. This could afford greater control over maintenance of self-renewal of embryonic stem (ES) cells while at the same time providing a convenient system to enable larger scale expansion of pluripotent ES cells for lineage-specific differentiation, which is a key requirement for translational research from bench to clinic. This proposal will investigate and develop the use of dynamic 3D bioreactor culture systems, with appropriate biomaterials, for the maintenance and expansion of pluripotent ES cells. The overall objectives are two-fold, first to investigate the ability of 3D scaffolds and culture systems to maintain self-renewal of murine ES cells and secondly, to extend this investigation to include studies on human ES cells. The specific project aims are: 1. To investigate the ability of different 3D scaffold materials to support murine ES cell self-renewal. 2. To examine the effect of surface treatment of scaffolds on murine ES cell self-renewal. 3. To compare the ability of different culture systems to influence murine ES cell self-renewal on 3D scaffolds. 4. To determine the effects of cytokines and small molecules on self-renewal of mES cells on 3D scaffolds. 5. To examine the ability of dynamic 3D culture systems to maintain pluripotency of human ES cells. Murine ES cells will be cultured in the presence of leukaemia inhibitory factor (LIF) to maintain the cells in an undifferentiated state. In later work we will use human ES cells (cultured either on a murine embryonic fibroblast feeder (MEF) layer or matrigel, supplemented with basic fibroblast growth factor and MEF conditioned media). A variety of extracellular matrix-like scaffold materials will be used in this research to support ES cells in an undifferentiated state. Initially, we will use gelatin-coated tissue culture plates and microcarriers for 2D and 3D culture respectively. Success in maintaining ES cells in an undifferentiated state as we move from 2D to 3D culture systems will allow us to investigate a wider range of scaffold materials typically used in tissue engineering research (e.g. collagen, and biodegradable polymers). We will also investigate the effects of different attachment factors on ES cell behaviour on the matrix-like scaffolds. In order to investigate ES cell behaviour in dynamic 3D culture, we will use several bioreactor culture systems that are in routine use at the University of Bath. These bioreactors are characterised by their ability to feed the cells continuously, to efficiently supply nutrients to support 3D cell and tissue culture, and allow flexibility in how they are operated so that the effects of key variables on ES cell behaviour can be investigated. Central to our studies will be the ability to quantitatively assess effects on ES cell self-renewal arising from propagation on the biomaterial scaffolds and in the different 3D culture systems. We will use a range of assessments for our studies including: cell proliferation assays, murine and human ES cell self-renewal assays, immunohistochemistry, immunoblotting, scanning electron and atomic force microscopy.