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Title: Magnetic targeting of stem cell therapies
Author: Harrison, Richard P.
ISNI:       0000 0004 5920 1691
Awarding Body: University of Nottingham
Current Institution: University of Nottingham
Date of Award: 2016
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Stem cells may offer solutions for many health issues facing the world’s population. Early Biotech-led approaches are supporting novel mesenchymal stem cell (MSC) therapies through biomedical trials. However, their potential benefits are currently curtailed by challenges linked to high cell dose requirements which pose availability and manufacturing challenges, combined to suboptimal delivery methodologies. Whilst systemic delivery may be suitable for many pharmaceuticals, more complex and selective treatments such as emerging cell therapies require smarter targeting strategies on safety and cost/benefit grounds. Several groups are developing targeting strategies to guide stem cells to specific locations and hold them in situ whilst performing a repair. The targeting approach presented here uses superparamagnetic iron oxide microparticles (MPs) loaded within stem cells to facilitate control of the cells using magnets. Magnetic resonance imaging (MRI) can be used to monitor the loaded cells’ contribution to the repair process. Questions remain around MP safety and effects on both delivered cell therapies and the receiving patient. Presented data demonstrates labelling of MSC populations with a commercially available MP called SiMAG in two sizes (500 nm and 1000 nm). Particles were assessed for characteristics which influence their suitability for labelling and were found to have a non-uniform variable structure and size. Labelling was found to be both rapid and effective with low 10 µg/Fe/mL labelling doses able to distinguish cell populations by flow cytometry. Super-resolution microscopy, fluorescent microscopy and transmission electron microscopy were used to determine the location of particles within the cell and were noted to accumulate around the nucleus in large vesicles. Uptake into the cell was found to be influenced by serum with 10% serum resulting in a 75% drop in relative uptake over a 24 hour period. Potential sharing of MP between MSCs was investigated both qualitatively with fluorescent microscopy and quantitatively in a MSC co-culture experiment. No statistically significant sharing of MPs between MSCs could be seen to be taking place between populations. The fate of MPs within MSCs was investigated using pH nanosensors to interrogate the internal cell pH. A novel flow cytometry assay using pH nanosensors and MPs was used to examine the internal pH of large populations of cells. This yielded results which suggest a pH decrease over 4 days from pH 5.5 to 4.7 followed an increase to 5.4 by day 6. This effect is suggested to be caused by a complex pH mediated degradation of MPs followed by increase in pH due to the degraded iron overloading the cell. This degradation was carried out in simulated lysosomal conditions and found to act in a similar way. Macrophages were also used to test degradation and again they were found to reduce the fluorecense of the MPs rapidly over 7 days. The ability of MSCs to tolerate MPs without impacting cell health was probed with a range of assays. The metabolic assay Presto blue demonstrated doses of 10 µg/Fe/mL did not impact the metabolic status of the cell. This was tested with other potential surface chemistries of the same particle design and these were also found to be well tolerated. Membrane intergrity was assessed with flow cytometry for both 500 and 1000 nm SiMAG and was found to have no damaging effects present at 10 µg/Fe/mL. SiMAG 1000 nm was found to have no membrane compromising effect all the way up to 100 µg/Fe/mL. Cell identity was assessed with common MSC markers established by the Dominici position paper and no change to expression was found to occur even with repeated, high dose long-term (14 day) labelling strategies. As particles accumulate round the nucleus, deleterious effects of MP on DNA were tested using the comet assay and visual inspection with no statistically significant increase up to the maximum tested of 100 µg/Fe/mL. Similarly, no effect on cell cycle status was noticed for populations of MSCs. The retention of cell “function” was tested not only following labelling, but following hypothermic storage of cells to simulate shipping to a clinical setting. This was carried out for a range of clinically relevant cell types including mesenchymal stem cells (MSCs), cardiomyocytes (CaM) and ReNeuron neural stem cells (ReN). MSCs were found to freely differentiate to tri-lineage osteogenic, chondrogenic and adipogenic lineages. CXCR4 expression was measured as a marker of MSCs ability to home in on damage and was found to be raised in response to MP presence. CaM were found to resume beating both after hypothermic storage, as well as at high (1000 µg/Fe/mL SiMAG doses). ReN cells were found to be more sensitive to SiMAG with only 10 µg/Fe/mL doses tolerated although successful neural differentiation was still possible. The ability to culture MSCs and label them in a scalable manufacturing scenario was also examined and found to be possible. SiMAG was demonstrated to be a suitable labelling agent both for imaging as well as magnetic manipulation. Precise magnetic manipulation of labelled cells was demonstrated both as an individual cell and as a cell population moving through a simulated tissue gel. Entrapment of labelled cells from a simulated circulatory system was also shown to be possible with close to 100% of cells recruited in the first pass. The fluorophores on SiMAG were not strong enough to be visualised on their own and quantum dots were used to demonstrate successful retention of labelled MSCs in an ex vivo rat model. MRI was however shown to be a suitable method for assessing the location of labelled cell populations at even low cell concentrations ~1x106 and low SiMAG doses of 5 µf/Fe/mL. In conclusion, this study indicates that MPs represent a cell-safe and effective potential tool to better target cells. The key benefit of this cell labelling technology is in the high degree of understanding over the entire labelling process from entry through to degradation. In addition, this labelling technology has been shown to be cell-safe in a large number of cells both for physical health and basic function. Whilst this should be further explored for further, more specific applications, it makes a compelling case for SiMAG as a multi-functional tool for cell manipulation and tracking.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QU Biochemistry