Innovative Approaches to Tracking of Mesenchymal Stem Cells: In Vivo Tracking of Mesenchymal Stem Cells Using 19F MRI

Abstract

Clinical translation of MSCs-based therapies remains challenging, and one of the key cause for this failure is the lack of tools to determine the fate of these transplanted cells non-invasively and longitudinally. Whatever the strategy, namely using either MSCs as direct effector cells or using genetically manipulated MSCs, it is imperative that the fate of these transplanted cells is understood to ascertain homing efficiency, dosing regimens, optimal timing and effective route of delivery to the diseased tissue. Hence, in vivo cell tracking is potentially a powerful tool to monitor non-invasively the distribution and accumulation of therapeutic cells such as MSCs. Development of accurate imaging modalities to track MSCs would allow clinicians to determine whether cell delivery has occurred at the appropriate site and if cells have reached their targeted location. Consequently, non-invasive real-time imaging techniques need to be developed to optimise and allow success of cell-based therapies. The absence of a confounding background signal and consequent unequivocal assignment makes 19F MRI one of the most attractive modalities for the tracking of injected cells in vivo. In Chapter 2, we evaluated the feasibility of poly(OEGMA-co-TFEA) as a potential 19F MR tracer for MSCs. Whilst these monomers contain a relatively low weight-fraction of fluorine, unique monomers with a higher fluorine content should be explored to achieve higher imaging sensitivity. In this chapter, we also describe the synthesis of novel partly-fluorinated polymeric nanoparticles with small size and high fluorine content as MRI agents. The polymers, constructed from perfluoropolyether methacrylate (PFPEMA) and oligo(ethylene glycol) methacrylate (OEGMA) have favourable cell uptake profiles and excellent MRI performance. To facilitate cell studies the polymer was further conjugated with a fluorescent dye creating a dual-modal imaging agent. The efficacy of labelling of MSCs was assessed using 19F NMR, flow cytometry and confocal microscopy. The labelling efficiency of 2.6 ± 0.1 x 1012 19F atoms per cell, and viability of >90 % demonstrates high uptake and good tolerance by the cells. This loading translates to a minimum 19F MRI detection sensitivity of ~ 7.4 x 103 cells/voxel. Additionally, in Chapter 3, our results demonstrate that PMSCs can be efficiently labelled with this new fluorinated copolymer, without the use of transfection agents, and with negligible deleterious effects on the viability, mitochondrial and phenotypic function of the cells. Chapter 3 also describes in vivo longitudinal detection and bio-distribution of labelled PMSCs in animal models using 19F MRI and optical imaging. In Chapter 4, we report a comprehensive evaluation to ascertain uptake mechanism involved in the internalisation in MSCs of our copolymer that was also effectively employed as a 19F MR tracer. Commonly used chemical inhibitors, which block specific pathways and endocytic markers, are utilized to ascertain the route of uptake in the cells. Furthermore, studies were conducted to identify co-localisation of copolymers with specific organelles, and endocytic markers used to validate mechanism of uptake adopted by these copolymers.Overall, these copolymer show outstanding potential for 19F MRI cellular tracking and for quantification of non-phagocytic and therapeutically relevant cells in vivo.