Whole body characterisation of bone marrow-derived cell kinetics: development of a syngeneic bone marrow chimera model for positron emission tomography with 18F-PBR111.

Abstract

Monitoring of cell transplantation in vivo is crucial to understanding how the progeny of stem cells contribute to or ameliorate disease. Although animal models enable researchers to understand the behaviour of specific cell types in great detail, they typically require invasive procedures or large numbers of animals in order to track sdisease progression or treatment efficacy over time. Positron emission tomography (PET) has emerged as a promising non-invasive method of tracking transplanted cells in preclinical research, as it enables researchers to track cells longitudinally over the lifespan of the host animal. Although existing reporter gene PET methodologies are under development, they are subject to a number of restrictions which limit their usefulness in preclinical research. These limitations include the need to transduce cells with reporter genes, which is inefficient and may lead to unwanted phenotypic changes in the cells of interest. Another major limitation for many applications is the inability of many reporter ligands to cross an intact blood-brain barrier. Described in this thesis is a novel methodology utilising the transplantation of wild-type bone marrow cells into mice lacking the Translocator Protein (18kDa; TSPO). The TSPO knockout mouse serves as a null-background model for transplantation of unmodified bone marrow cells which express endogenous TSPO. These TSPO-expressing cells are then targeted for PET imaging by a TSPO-specific radio-ligand, 18F-PBR111. PBR111 binds selectively to TSPO and can penetrate the intact blood-brain barrier, making it a promising approach for overcoming some of the limitations of reporter gene imaging. In brief, TSPO-knockout mice (and wild-type controls) were conditioned with a sub-lethal dose of gamma radiation and transplanted with nucleated bone marrow cells from C57BL/6 mice engineered to express green fluorescent protein (GFP). At various stages of engraftment ranging from 1 to 12 months post-transplant, mice were anaesthetised with 1-4% isoflurane, cannulated via the tail vein, and injected with 0.2nM of 18F-PBR111. Data was collected with an Inveon pre-clinical PET/CT camera for 50 minutes post-injection. Images were re-constructed into 20 time frames and were again re-constructed using a 3-dimensional ordered subset expectation maximization algorithm (3D-OSEM). From this, time activity curves (TACs) were extracted from regions of interest drawn on co-registered PET/CT images, corresponding to whole organs. TACs were then used to calculate standardised uptake values for each region of interest. Sections taken from collected tissues were later incubated with 125I-CLINDE in order to visualise receptor density in tissues independently of pharmacokinetic parameters associated with IV tracer injection. Sections were also stained with a monoclonal TSPO antibody in order to identify the TSPO-expressing cells at high spatial resolution. Engraftment rates in blood were assessed at 1, 3 and 6 months post transplantation using quantitative real-time PCR in order to determine whether haematopoietic reconstitution in TSPO knockout mice resembles its wild-type counterparts, which is important for establishing whether the results observed in vivo are physiologically ‘normal’ and therefore applicable to models of disease. Although engraftment rates were equivalent in wild-type and TSPO-/- background recipient mice 1 and 3 months post-transplantation, at 6 months engraftment rates in the TSPO-/- mice were significantly higher. Nonetheless, both genotypes displayed persistent engraftment of donor cells at a minimum of 6 months post-transplant, and observed up to 12 months post-transplant in the TSPO-/- recipients. This engraftment was detectable using both in vitro and in vivo methods (PET) in spleen tissue throughout the duration of the study, and in some tissues susceptible to inflammation such as lung and salivary gland at 6 months post-transplant. A pilot of longitudinal imaging also suggested this persistent migration of donor-derived cells could be detected up to at least half of the animal’s lifespan. Although some major successes in longitudinal monitoring of TSPO-expressing donor-derived cells was observed in this study, there were some notable exceptions, such as the bone marrow itself, susceptible to major spillover associated with free fluorine accumulating within bone, and regions marked by low level engraftment of cells, including brain, and lung parenchyma. For each tissue of interest, an in-depth discussion of the implications of the findings are presented, followed by an outline of the methodological challenges associated with whole-body imaging of rodents in this context. Overall, the model presented in this study has the potential for adaptation to a variety of models incorporating cell-based diseases or treatments, with the ability to track the accumulation of inflammatory cells in a variety of organs.