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The research reported in this thesis focused on the potential of neural precursor cells to provide a suitable source of neurones which can be used in cell replacement strategies for Huntington's disease. Specifically, the parameters affecting the differentiation of these cells into neuronal phenotypes were addressed and increasing the survival of proliferating and differentiating neurones was attempted. In vivo characteristics and the fibre projections of primary and 10 day expanded ENPs was also assessed. The limitations of xenografts in this thesis led to the search for an alternative model system for such experiments. Chapter Three involved an extensive study investigating the effects of a range of concentrations of FGF-2 and EGF on the proliferation and more importantly the neuronal differentiation of murine ENPs over 6 passages in culture, and it was found that the concentration had an effect on the neuronal proportion as well as the neuronal yield of these cultures. Chapter 4 examined the turnover of neuronal precursors in the ENP population cultured in the presence of FGF2 and EGF, using BrdU. The ongoing proliferation of neuronal precursors within ENP cultures was observed and the addition of the growth factors: CNTF, BDNF, HGF and NGF, to enhance the survival of these neurons on differentiation had no effect. Chapter 5 examined the potential of 10 day expanded human striatal ENPs to maintain a striatal like phenotype both in vitro and in vivo in comparison to primary foetal tissue. In vitro after 10 days expansion ENPs differentiated into DARPP-32 positive neurons and this characteristic was maintained in vivo, in a lesion model of HD, albeit to a much lesser extent. This study was limited by the need for ongoing immunosuppression which reduced the life span of the host animal. Chapter 6 investigates further the potential of ENPs. The ability for these cells to send long projections in the host brain and therefore repairing the circuitry lost or damaged as a result of the disease. A four way analysis was carried out examining both alio- and xenograft environments with both primary and 10 day expanded ENPs. Mouse grafts were used to address the allograft environment given that such an experiment is not possible with human tissue and both human and mouse tissue addressed the xenograft environment. To overcome the issues associated with labelling the grafted tissue in the host brain, several techniques were employed, including the use of the GFP transgenic mouse, lentiviral labelling of the cells with the LacZ gene and iontophoretic labelling of the graft with anterograde tracers. ENP grafts were shown to send out longer projections than that of primary tissue although this may be due to migration of the grafted cells. Chapter 7 addresses the issue of immunosuppression of xenografted animals. An alternative model system was explored with the hypothesis being that it would be possible to tolerise the animal in the neonatal period to the xenograft tissue that would subsequently be used for intrastriatal grafting in the adult. Indeed, tolerising the animal resulted in healthy surviving grafts in the adult without the need for daily immunosuppression. Work presented in this thesis contributes some understanding to the biology of neural stem cells and neural xenografts that may ultimately be used for neural transplantation therapies in HD. |
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