Tools for evaluating safety and cell fate
Key safety concerns with cell-based regenerative medicine therapies include the risk that the administered cells could cause intra-vascular thrombosis, form tumours or mal-differentiate1. To evaluate safety, it is necessary to establish the whole-body and intra-organ biodistribution of the cells, initially in un-injured models, and then in injury models. This will be achieved by developing non-invasive multi-modal imaging strategies, including magnetic resonance imaging (MRI), for the detection of cells labelled with MR contrast agents (superparamagnetic iron oxide nanoparticles; SPIONs); bioluminescence imaging (BLI) for the detection of cells constitutively expressing luciferase reporters; the emerging technology, MSOT, developed by iThera, to detect cells labelled with optoacoustic contrast agents (gold nanorods; GNRs) and positron emission tomography (PET) using radionuclides.
Innovative multi-modal imaging is necessary to overcome the limitations of the different technologies. MRI offers excellent spatial resolution, but temporal resolution is poor. Thus, MRI can monitor intra-organ biodistribution, but whole-body imaging is not feasible. BLI has poor spatial resolution, but its excellent sensitivity makes it ideal for monitoring whole-body distribution.In addition, BLI shows signal from only viable cells, whereas signals from nanoprobes can sometimes persist even if cells are no longer viable. MSOT overcomes many of the limitations of MRI and BLI; for instance, it has excellent sensitivity, good spatial and temporal resolution, accompanied with multiplexing and functional imaging capability2. Nuclear imaging, on the other hand, has excellent sensitivity but the signal is short lived. State-of-the-art imaging facilities at the University of Liverpool will enable the combination of imaging probes and technologies so that the advantages of each modality is fully explored, providing a complete picture of the cellular biodistribution.
To ensure spatio-temporal complementarity of the data obtained from the different modalities, we will use develop data fusion tools to facilitate multimodal data integration. These innovative multi-modal imaging strategies, which go beyond the current state-of-the-art, will enable us to gain in-depth knowledge of the behaviour of transplanted cells to allow a comprehensive efficacy evaluation of the different types of MSCs and their derived products.
Tools for evaluating efficacy and injury progression
State-of-the-art MRI techniques, all of which are clinically translatable, will be employed by the University of Liverpool to assess changes in kidney morphology. These include 3D rendering to assess organ volume changes, and perfusion imaging with dynamic contrast enhanced imaging (DCE-MRI).
With iThera, we will devise MSOT imaging applications for assessing efficacy. We have shown that MSOT can be used to visualise the renal clearance of the NIR dye IRDye 800 carboxylate and can predict the extent of histological damage in a mouse model of kidney injury2. However, this dye is not ideal for monitoring renal function because it displays a high level of plasma protein binding. Therefore, the novel NIR tracers developed in WP1 for use in the diagnostic device will be evaluated for their ability to monitor renal function using MSOT.
At the study end-points, we will apply novel ex vivo imaging technologies based on recently described tissue clearing reagents and protocols4 that enable intact organs to be analysed microscopically by making them transparent. In some experiments this will be combined with a recently described tissue expansion technique that can improve imaging resolution 5-fold5. This approach has several advantages over traditional histological methods; for instance, antibody diffusion through intact adult mouse kidneys can easily be achieved so that when used in combination with 2-photon or light sheet fluorescence microscopy (LSFM), the whole kidney can be imaged. This circumvents the problems of bias that can arise when the histological sections selected for analysis are not representative of the whole organ. By using appropriate antibodies, this new technique will permit (i) the location of individual MSCs to be mapped within the specific organs; (ii) the extent of fibrosis to be determined; (iii) the extent of vascular rarefaction; and (iv) the number and size of glomeruli to be counted.
1. Heslop et al (2015) Stem Cells Trans Med 4: 389-400
2. Sharkey et al (2016) Eur J Pharmacol 790:74–82
3. Scarfe et al (2015) Sci Rep 5:13601
4. Klingberg et al (2018) J Am Soc Nephrol 28:452-9
5. Unnersjo-Jess et al (2018) Kid Int 4 , 1008 – 1013