Pope, Christopher S.; (2024) Imaging and Therapy of Stem Cell Derived Extracellular Vesicles in a Mouse Model of Cardiac Ischaemia. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human trials have shown stem cell treatment can improve cardiac function in heart failure patients however these findings are inconsistent. Comparable benefit from stem cell-conditioned media has opened the field to a paracrine signalling means of cardioprotection. Extracellular vesicles (EVs) are a promising effector of this: being identified in stem cell media, inducing survival upregulation to cells, and pertaining in vivo protection in animal models. How exactly they mediate such benefit and where they go or stay, however, remains elusive. In this work I have scaled up ultracentrifuge-based isolation of small EVs (sEVs, i.e. <200nm in size)from a mouse mesenchymal stem cell line (mMSC) and characterised these by nanoparticle tracking analysis (NTA). To image & track these vesicles in vivo I transfected my cells to express an sEV-localised reporter enzyme for bioluminescent imaging (BLI), as well as labelling with a fluorescent dye and radiotracer. To test their benefit in vitro, I administered sEVs to infarct-challenged cardiac and non-cardiac cell lines. I optimised an animal model of myocardial infarction (MI), validating this with noninvasive cardiac imaging using preclinical MRI and ultrasound technologies. I tested the in vivo benefit of sEVs over a saline control when delivered intramyocardially at the site and time of ischaemia via subsequent serial assessment of cardiac function and infarct size. My results showed I obtained 100 to 140 nm-sized small EVs of high purity from my mMSCs, but my repeated attempts to prepare these for in vivo image-tracking were unsuccessful. In vitro therapy to cells proved negative. In vivo study, however, identified significant, continued improvement in cardiac function and tissue measured by Cine & LGE MRI, specifically localised to the region of sEV delivery from one day to two weeks postsurgery. This was supported by multi-slice ultrasound data. These findings demonstrate the administration of mMSC-derived sEVs was cardioprotective in this region and that this early benefit was sustained. This work adds to the body of research of cardioprotective stem cell paracrine signalling in vivo and supports that this is in part directed by released sEVs. More in vivo imaging is needed though to fully describe extracellular vesicle behaviour in vivo and how this mechanism of protection is imparted.

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