Medicine (Austin & Northern Health) - Theses
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ItemHuman in vitro models of ischaemic stroke: a test bed for translation( 2013)Over 500 therapeutic agents have shown protection against ischaemic injury in animal models of stroke. However, efficacy has yet to be successfully translated into humans. The aim of this thesis is to examine the potential uses of stem cells to improve stroke therapy. Systematic review and meta-analysis of stem cell therapy for stroke in animals revealed that while there was an overall benefit, much of the heterogeneity in the data was accounted for by facets of stroke modelling rather than stem cell biology. Analysis of the data for spinal cord injury, which shares key pathophysiological mechanisms with stroke, revealed the same phenomenon. My conclusion from this work is that we do not fully understand what determines outcome in animals and should be cautious before applying this incomplete knowledge to humans. An alternative use of stem cells is to generate human neurons (and other central nervous system cell types) as a test-bed for drug screening. Analysis of the literature revealed that this hypothesis was untested and might provide the quickest route to practical use of stem cells. The experimental aims of this thesis were therefore to differentiate human embryonic stem cell (hESC) lines into neurons, develop models of ischaemic injury and test potential therapeutic agents. Induction of neuronal differentiation of hESCs was achieved by using the bone morphogenic inhibitor protein, Noggin. The resulting progenitors were then grown in the presence of EGF and bFGF until they formed neurospheres. Removal of the growth factors allowed the neurospheres to differentiate into neurons which were then cultured for 11 days prior to injury. Two methods of injury were used: (i) oxygen glucose deprivation (OGD) and (ii) H2O2 induced oxidative stress. Four potential therapeutic agents (hypothermia to 33°C, melatonin at the concentration range of 10nM to 1mM, VAS2870 at 10µM to 50µM and NXY-059 at 1µM to1mM) were tested and cell death was quantified using a lactate dehydrogenase (LDH) assay and TUNEL staining. Hypothermia reduced H2O2 and OGD induced cell death by 53% and 45% respectively at 24 hours. The neuroprotective effect of hypothermia diminished with time however, it was neuroprotective even when administered six hours after H2O2 induced cell death. 100µM Melatonin and 50µVAS2870 showed the highest neuroprotective effect and both drugs reduce H2O2 and OGD induced cell death by >60% at 24 hours. The neuroprotective effect of both melatonin and VAS2870 diminished with time and, unlike hypothermia, they had no effect on cell death when administered six hours after injury induction. NXY-059 provided no effect on neuronal cell survival in any of the injury models. This thesis provides the first investigation of protection of embryonic stem cell derived neurons by four different potential therapeutic agents. While hypothermia, melatonin and VAS2870 which are still under consideration for clinical trial show promising activity, NXY-059 which failed in the clinic, does not. These results suggest that human embryonic stem cells have a potential to be differentiated into different populations of functional, mature, synaptically active neurons, and could provide a useful drug screening tool.