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Title: Mms6 expression in adipose-derived mesenchymal stem cells : development of biogenic magnetic nanoparticles and its therapeutic potential
Author: Kerans, Fransiscus Fiano Anthony
ISNI:       0000 0004 9349 2568
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2020
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Magnetic nanoparticles (MNP) are attracting interest for a range of biomedical applications being used either alone or as part of cell-based therapies. An area of particular interest is magnetic nanoparticle-mediated hyperthermia (MNHT), when MNP absorb energy from alternating magnetic fields (AMF) and transform this energy into heat which results in cancer cell death. While promising, the use of MNP for diagnosis and therapy has been limited by their rapid removal from the blood and biological barriers at the tissue and cellular levels. Moreover, MNP may have adverse side effects when used clinically. To overcome these problems there is increasing interest in the development of cellbased strategies to deliver MNP. Current strategies include combining commercially available MNP with mesenchymal stem cells (MSC), as these cells can migrate to sites of tissue injury and tumor growth. However, problems with MNP cytotoxicity have hindered progress in this area and need to be overcome. Therefore, the ultimate aim of this PhD project was to find an alternative way to develop MSC that contain MNP using a genetic engineering approach by which the cells can be induced to stably produce biogenic MNP and to establish whether such an approach could be of value for MNHT for cancer treatment. To achieve this goal, the experiments conducted in this PhD project involved transfection of adipose-derived mesenchymal stem cells (AD-MSC) to introduce a synthetic magnetic gene, mms6, into the cells. The gene is originally derived from Magnetospirillum magneticum AMB-1, a genus of magnetotactic bacteria (MTB), which has unique intracellular structures called magnetosomes. The magnetosomes contain iron-rich magnetic nanoparticles that are enclosed within a lipid bilayer membrane. During the formation of magnetosome, mms6 has been known for its key role in the formation of uniform isomorphic magnetite nano-crystals and helps regulate the crystal morphology of magnetite. Due to the unique feature of this gene, therefore the novelty of this study was in introducing codon-optimised mms6 into AD-MSC, enabling the cells to produce biogenic nanoparticles. In this study, mms6 mRNA expression in AD-MSC, following transfection, was demonstrated by reverse transcription polymerase chain reaction (RT-PCR). The HisGFP tag Mms6 protein expression was demonstrated by Flow cytometry, Western blot and GFP imaging analysis revealing the expression of Mms6 protein in AD-MSC. Furthermore, for stable mms6 expression, a lentiviral transduction approach was used. AD-MSC stably expressing mms6 were then used in in vitro MNHT studies. The effect of mms6 stable expression on MSC markers of stemness and differentiation ability of AD-MSC were also investigated. The cellular ultrastructure of AD-MSC expressing mms6 was demonstrated by transmission electron microscopy (TEM), revealing the presence of nanoparticles. The magnetism of AD-MSC expressing mms6 was proved by superconducting quantum interference device (SQUID). Furthermore, as a comparison study, Ferucarbotran, chemically synthesized superparamagnetic nanoparticles, were also used to magnetize AD-MSC. Both AD-MSC expressing mms6 and Ferucarbotran-loaded AD-MSC were used in in vitro MH and magnetic resonance (MR) imaging studies. In vitro studies of MNHT were undertaken to investigate whether AD-MSC expressing mms6 and Ferucarbotran-loaded AD-MSC could have a MNHT effect when exposed to an AMF. Cell viability, cell apoptosis and HSP70 expression were assessed to investigate the MNHT effect. The results did not indicate that the AMF application on AD-MSC expressing mms6 have a MNHT effect, showing no observable difference in cell viability, cell apoptosis and HSP70 expression. In vitro MRI experiments were conducted to test whether mms6 can function as a MR reporter gene for molecular imaging. The result revealed AD-MSC expressing mms6 produced a detectable magnetic signal, revealing a promising potential of mms6 as a reporter gene for MR imaging. Overall, the results indicate that an MTB gene, mms6, can be expressed in AD-MSC without an adverse effect on important cell functions. Moreover, these results also indicate that no appreciable cell necrosis or cell apoptosis was found when AD-MSC expressing mms6 were exposed under AMF. However, this study has helped our knowledge on the biosynthesis of MNP in AD-MSC which also has potential use in MR imaging.
Supervisor: Salter, Donald ; Boyd, Chris Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: mesenchymal stem cells ; magnetic nanoparticles ; magnetotactic bacteria ; mms6 gene transfection ; AD-MSC