Tissue engineering of any tissue type requires the combination of a supporting scaffold, a range of biological factors and a suitable source of cells. This source of cells must satisfy a number of criteria:- • The ability to form all of the mature/specialised cell types found in the target tissue. • Readily obtainable. • Readily maintainable in the laboratory without requiring excessive resources or time. For intestinal tissue engineering there are a number of issues associated with the use of tissue derived stem cells particularly quantity of normal tissue available (from the patient) and maintenance and expansion of the cells when cultured in vitro. Using embryonic stem cells offers a potential alternative strategy but methods must be established to efficiently differentiate the cells towards the desired fate. Many strategies for differentiating embryonic stem cells are based upon treatment with growth factors in vitro. There is a (logistical) limit to the degree of complexity that these systems can achieve and therefore a limit to the number of differentiation signals that occur during in vivo development that can be mimicked. In recent years using embryonic tissue to provide signals to undifferentiated cells has proved a successful method of directing the differentiation of naïve cells towards a particular fate (with the choice of tissue determined by the desired target cell type). The aims of this thesis were to explore the potential of differentiating embryonic stem cells towards the intestinal progenitor fate using a combination of in vitro cell culture treatment with the growth factor Activin-A and ex vivo co-culture with embryonic chick gut tissue. Previous studies [Kubo et al 2004, Tada et al 2005, Yasunaga et al 2005, D’Amour et al 2005, MacClean et al 2007] have shown that Activin-A treatment will induce embryonic cells to more efficiently differentiate to definitive endoderm, the germ layer from which the intestines (and other visceral organs) arise. These techniques were applied to the Columnar Epithelial Epiblast murine embryonic stem cell line and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. Activin-A treatment produced an upregulation of definitive endoderm markers at both the mRNA and proteomic levels compared to the control conditions. However the cell population produced retained expression of pluripotent markers and showed some expression of markers of other cell lineages. Further studies [Sugie et al 2005, Fair et al 2003, Van Vranken et al 2005, Coleman et al 2007, Krassowska et al 2006] have shown that co-culture of embryonic stem cells with early stage embryonic tissue can induce the formation of particular tissue types; the tissue must be selected based on proximity to the target cell type during development. This exposes the embryonic stem cells to the signals that prompt differentiation towards the target tissue during normal development. With gut tissue much signalling occurs between the different tissue layers that make up the whole organ both during development and in adult tissue. Ex vivo co-culture of murine embryonic stem cells with embryonic chick gut tissue was used to direct their differentiation to the intestinal epithelial stem cell fate. Before the co-culture was carried out various experiments were carried out to establish if the proposed protocol was viable e.g. defining how long chick gut tissue explants could survive in culture. Once this had been established co-culture experiments were undertaken and cell differentiation was then evaluated at the molecular level using Reverse Transcription-Polymerase Chain Reaction, immunocytochemistry and Western blotting. The cells showed some expression of intestinal epithelial stem cell markers at both the mRNA and proteomic levels following co-culture. The cells were also assessed at a physiological/functional level by evaluating their ability to form a functional intestinal epithelial barrier. This was achieved using an in vitro co-culture model with intestinal subepithelial myofibroblasts by measuring transepithelial resistance, permeability to protein and morphology in a simple tissue model co-culture. The cells did not display the morphological or physiological characteristics associated with intestinal epithelial cells in the model system. Overall this work has shown that co-culturing pluripotent mES cells with embryonic chick gut tissue can induce differentiation towards the ISC fate. Pre-treating the cells with growth factors in vitro did not seem to enhance this differentiation but there was scope to refine these techniques. Following the differentiation protocols the cells did not display the desired physiological characteristics but again there was scope to refine the techniques particularly with regard to selecting cells positive for the expression of the chosen molecular markers. These techniques show promise but do require some further development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:559695 |
| Date | January 2012 |
| Creators | Carter, David Andrew |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/12713/ |
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