Development and appraisal of MRI contrast agents for the in vivo analysis of stem cell grafts

Williams, Susannah Jane 2012. Development and appraisal of MRI contrast agents for the in vivo analysis of stem cell grafts. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

The work reported in this thesis deals with the need for efficient in vivo tracking and monitoring of grafted cells and their subsequent survival. It is considered in the context of cell transplantation for neurodegenerative disorders, particularly Huntington’s and Parkinson’s disease, although the scope for a good contrast agent to monitor cells in vivo goes far beyond this. There is currently no routine method used to follow cells in vivo and it is crucial for the advancement of cell transplantation studies, both in terms of providing powerful longitudinal analysis and for decreasing the number of animals necessary per experiment. MRI allows good contrast resolution and provides details on soft tissue anatomy without being harmful to the subject. By labeling cells with an MRI contrast agent the labeled cells can be distinguished from the surroundings and information on location is attained. A good MRI contrast agent can provide more information than this though, but to date there hasn’t been an MRI contrast agent developed that can simultaneously provide good signal and be reflective of the graft changes, while not affecting the cell’s viability. The feasibility for in vivo MRI scanning of three MRI contrast agents were tested and detailed below. In Chapter 3 we looked to utilise SPIOs as contrast agents. Since SPIOs are the most widely used of contrast agents they were tested in mouse ES cells, expanded whole ganglionic eminence and rat ventral mesencephalon and successfully labeled all cell types. Problems were discovered in reference to the needle track leaving an MRI visible track that eclipsed the area of graft deposition, and while SPIOs did not hamper graft survival, only large grafts extending out from the needle track could be reliably measured. Chapter 4 examined the generation of ferritin constructs for use as contrast agents. Transgenes based on the ferritin subunits provide MRI contrast by increasing the iron content of a cell. Both subunits, heavy and light, were transfected into mouse ES cells and expressed to improve signal compared to overexpressing the ferritin heavy transgene alone, which has been done in the literature. The expected change in T2 relaxation compared to control cell lines was observed in vitro. In Chapter 5 the applicability for in vivo use of Chemical Exchange Saturation Transfer (CEST) was tested. CEST involves the selective saturation of protons of particular compounds that are then indirectly detected through the water signal. Current 2D RARE scans to pick up CEST are slow and not really transferable to animal studies, due to the large increase in scan time for each extra image slice required, here alternative 3D FLASH scans were developed that still allowed the CEST contrast change to be observed over a whole sample in a reasonable time frame. A transgene based on CEST was tested in vivo and expression was lost even under antibiotic selection. Work in this thesis contributes some understanding towards the promises and pitfalls of three MRI contrast agents, two of which may ultimately be used more routinely for cell tracking in the future.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General) Q Science > QH Natural history > QH301 Biology
Funders:	BBSRC
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:13
URI:	https://orca.cardiff.ac.uk/id/eprint/43157

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