Cooke, Michael John (2009) The influence of the extracellular matrix on cell behaviour. Doctoral thesis, Durham University.
Stem cell research has raised great interest in the scientific community as it has the potential to form multiple cell types and it is believed they hold the key to curing many diseases. However, there is a need for better understanding of how to control these cells and further research investigating methods for controlling and directing these cells is required. Pluripotent stem cells transplanted into immune-deficient mice 'spontaneously’ differentiate and proliferate to form a complex mass of differentiated and undifferentiated cells, teratomas - teratoma assay. Such tumours are generally haphazard in their organisation however; they do contain some structures similar to those observed in the embryo. Teratoma formation is a useful model to explore the developmental potential of stem cells and study aspects of tissue development. Examination of how the anatomical location into which human pluripotent stem cells are grafted influences their growth in situ allows investigation of how these cells are affected by different areas within the body: cells grafted into the liver rapidly produce large tumours containing predominantly immature cells whereas, subcutaneous implants were significantly slower growing and formed tumours composed of differentiated tissues. These different growth patterns indicate how environmental cues within the niche affect stem cell behaviour. One factor which contributes to the maintenance of a niche is the extracellular matrix (ECM). To investigate how endogenous ECM affects teratoma behaviour, co-transplantation is carried out with stem cells and ECM components. The ECM extract Matrigel™ dramatically increased the success rate of teratoma formation and size with no detectable affect on teratoma composition when compared to controls and removal of the growth factors from the co-transplanted ECM extract had no effect on teratoma success rate, growth rate, or composition. To study the effects of the ECM in vitro, components of the ECM are often used topcoat glass or plastic surfaces to enhance cell attachment in vitro. Fragments of ECM molecules can be immobilised on surfaces in order to mimic the effects seen by whole molecules. In this study a novel technology developed by Oria Protein Technologies for the immobilisation of peptide sequences from ECM proteins is evaluated. By examining: the adherence of cultured PC 12; neurite outgrowth from PC 12 cells; and neuronal differentiation of neural stem/progenitor cells (NSPCs) it is shown that peptides from collagen I, collagen IV, fibronectin and laminin can mimic surfaces coated with ECM proteins. Collectively, this data demonstrates that peptides from ECM proteins can be immobilised in functional fashion to control cell behaviour. Surfaces with adsorbed proteins and biomimetic surfaces presenting peptide motifs from ECM proteins are used to investigate and explain observations from in vivo teratoma experiments. In vivo, Matrigel™ increases the gene expression of the pluripotent stem cell marker Oct4, increasing the pluripotent cell percentage and thus increases the likelihood of teratoma formation. In vitro, Matrigel™ also increases the gene expression of the proliferative marker Ki67, indicating that large teratomas from by the co-transplantation of stem cells with Matrigel™ could be due to increased cell proliferation.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||08 Sep 2011 18:24|