Dobson, R.; (2010) Tracking endogenous and grafted neural progenitor cells in normal and ischaemic brains using MRI contrast agents and genetic labelling. Doctoral thesis , UCL (University College London).

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Abstract

Cerebral ischaemia is a major cause of mortality and morbidity globally. Neural stem and progenitor cells (NPC) have the potential to contribute to brain repair and regeneration after an ischaemic event. Both endogenous and grafted NPC have been shown to migrate towards the ischaemic lesion, and differentiate into neurons. This thesis investigates methods of labeling and tracking the migration neural progenitor cells to a site of cerebral ischaemic injury, using magnetic resonance imaging (MRI) contrast agents and transgenic lineage tracing techniques. First, labeling of exogenous NPC populations was investigated, for use in cell tracking in grafting studies. Cell labeling was optimized in vitro with fetal NPC using the iron oxide-based MRI contrast agent. A labeling method was developed using the FePro contrast agent, which maximized iron oxide uptake, was non-toxic to NPC, and did not interfere with NPC proliferation and differentiation. Labelled cells were then grafted into the brain after cerebral ischaemia, and imaged over four weeks using MRI. NPC migration was not observed in vivo, but an endogenous contrast evolved over time within the lesioned tissue, which presented a source of confounding signal for cell tracking. Endogenous ferric iron was observed in the lesion on histological sections. Several limitations of using MRI-based iron oxide contrast agents were highlighted in this study. To circumvent these limitations, we considered the development of gadolinium-based MRI contrast agents for cellular labeling and tracking, in collaboration with Imperial College chemistry department. Polymeric Gd-DOTA chelates were synthesized and designed for maximal r1 relaxivity, and their relaxivity and effects on cell viability were assessed. Through this approach, we identified a number of candidate polymeric Gd-DOTA chelates with high relaxivity and low cytotoxicity for use in cellular imaging and tracking studies. Next, cell tracking of endogenous NPC was investigated, using MRI contrast agent and transgenic lineage tracing approaches. A method of in situ labeling of endogenous NPC with the MRI contrast agent FePro was developed. NPC were labeled with FePro in situ, and their normal migration to the olfactory bulb, where they contribute to neurogenesis, could be imaged in vivo and ex vivo. In a second study, the migration of NPC constitutively expressing green fluorescent protein (GPF) under the promoters of genes of two developmentally distinct cortical and striatal NPC populations, was investigated following cerebral ischaemia. Both cortical and striatal populations of NPC were observed to contribute to the migrating streams of NPC that were observed in the striatum after five weeks post-ischaemia. These studies demonstrate that MRI contrast agents offer the potential for in vivo, longitudinal tracking of NPC migration, in both grafted and endogenous NPC populations. Coupled with transgenic lineage tracing, and used in animal models of CNS injury such as cerebral ischaemia, labeling and tracking the migration of NSC with MRI contrast agents can contribute to our understanding of NPC biology in pathological environments.

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