Dual-modality gene reporter for in vivo imaging

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T1-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd(3+) ion for the radionuclide, (111)In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging

Tracking Neural Progenitor Cell Migration in the Rodent Brain Using Magnetic Resonance Imaging

The study of neurogenesis and neural progenitor cells (NPCs) is important across the biomedical spectrum, from learning about normal brain development and studying disease to engineering new strategies in regenerative medicine. In adult mammals, NPCs proliferate in two main areas of the brain, the subventricular zone (SVZ) and the subgranular zone, and continue to migrate even after neurogenesis has ceased within the rest of the brain. In healthy animals, NPCs migrate along the rostral migratory stream (RMS) from the SVZ to the olfactory bulb, and in diseased animals, NPCs migrate toward lesions such as stroke and tumors. Here we review how MRI-based cell tracking using iron oxide particles can be used to monitor and quantify NPC migration in the intact rodent brain, in a serial and relatively non-invasive fashion. NPCs can either be labeled directly in situ by injecting particles into the lateral ventricle or RMS, where NPCs can take up particles, or cells can be harvested and labeled in vitro, then injected into the brain. For in situ labeling experiments, the particle type, injection site, and image analysis methods have been optimized and cell migration toward stroke and multiple sclerosis lesions has been investigated. Delivery of labeled exogenous NPCs has allowed imaging of cell migration toward more sites of neuropathology, which may enable new diagnostic and therapeutic opportunities for as-of-yet untreatable neurological diseases

Diffusion Tensor Imaging of the Central Nervous System Following an Injury to the Spinal Cord and Cell Transplant

The purpose of this dissertation research was to characterize the use of magnetic resonance diffusion tensor imaging (DTI) as a diagnostic and prognostic tool in understanding the changes that occur throughout the spinal cord and brain following a spinal cord injury (SCI) and following stem cell transplant. The diffusion of water inside the nervous system is dramatically altered around the lesion site following a traumatic SCI. However, following damage to the spinal cord, little is known about the diffusion characteristics away from an injury and even less is understood about DTI\u27s sensitivity to structural changes that occur following regenerative transplant therapies. The non-invasive nature of DTI could potentially allow for diagnostic and prognostic indicators of an SCI remote from injury and could provide physicians a method for tracking and monitoring the effectiveness of injected stem cells. To evaluate the sensitivity of DTI to structural changes in the central nervous system (CNS) following a traumatic SCI, diffusion metrics in the brain and cervical spinal cord were compared for four different injury severities in a thoracic contusion model of a rat SCI. Structural changes in the cervical region of the spinal cord after transplantation of C17.2 neuronal stem cells were also examined with the use of DTI. The findings from this dissertation suggest that diffusion tensor imaging is sensitive to changes in tissue structure in regions remote from injury and for cellular environments that increased astrocytic sprouting as a result of stem cell transplant. Mean water diffusion in the distal locations of the spinal cord and in the brain decreased following SCI. Neuronal stem cells that are known to elicit astrocytic proliferation produced mean increases in water diffusion. These results further clarify the potential for DTI to provide physicians a method to non-invasively monitor how the CNS changes following SCI and detec

Magnetic reporter genes for MRI-based stem cell tracking

Introduction: Over the past decades, several labelling techniques have been used in an attempt to track stem cells using magnetic resonance imaging (MRI). However, very few of these were able to definitely determine the precise location of stem cells within a living organism and monitor throughout a long term period, without loss or diffusion of the signal. A novel MRI cell tracking method described in 2005 proposed that reporter genes that could effectively increase the iron content of a target cell would allow a stronger contrast when imaged via MR. Being a fundamental part of the iron metabolism, transferrin receptor 1 (TfR 1) and ferritin heavy chain 1 (Fth 1) were naturally suggested to have the potential to increase the iron load of cells when overexpressed. More recently, there has been some interest in the reporter gene MagA, which is a known iron transporter found in magnetotactic bacteria. Aim: To evaluate the suitability of using TfR 1, Fth 1 and MagA as potential magnetic reporter genes for MRI-based cell tracking. Methods: Several cell and stem cell lines were transduced with a 2nd generation HIV-based lentiviral system containing one or more magnetic reporters. Viral transduction resulted in genome incorporation of bicistronic construct(s) with TfR 1 gene alongside a gene encoding a green fluorescent reporter (GFP) and/or Fth 1 and MagA gene alongside a red fluorescent reporter (RFP). This allowed for identification and monitoring of positive cells with complementing imaging modalities: MRI and fluorescence based methods. Transgenes were evaluated for integration stability over passages and their influence on iron homeostasis was assessed; also, integration and/or overexpression were confirmed at the mRNA and protein level. Finally, the influence of magnetic reporters on intracellular iron retention and MRI contrast capacity was tested both in vitro and in a model organism, the chick embryo. Results: After analysing all three potential magnetic reporters, TfR 1 was found to be the most promising, as its overexpression induced an adjustment of iron homeostasis in Chinese hamster ovary K1 cells, leading to higher intracellular iron accumulation relative to controls. The same adjustment was found in mouse mesenchymal stem cells (mMSC), but only when TfR 1 was overexpressed in conjunction with Fth 1, also leading to an increase in iron retention capacity. However, a limitation was found when overexpressing Fth 1 in mMSC, as permanent iron supplementation was needed in order to keep these cells viable. In contrast with previous studies, MagA gene integration posed some restrictions in certain cell lines studied. The results presented here show that while some cell types are able to stably maintain MagA expression over several passages, others fail to survive and die shortly after transduction, suggesting that a potential toxic effect may be originating from MagA gene integration. From the surviving cells, two were compared side by side and contradictory results were obtained, demonstrating that MagA would only be a suitable magnetic reporter for some cell types. Conclusion: The results obtained with this project are of relevance for reporter gene-based MRI cell tracking as they show that no single magnetic reporter is capable of generating detectable MRI contrast for a global cohort of cell lines. On the contrary, overexpression of endogenous genes or integration of foreign genes should be performed with caution and analysed on a case by case basis. Finally, for some cell type and magnetic reporter gene combinations, this study suggests that MRI could be a promising method for the longitudinal monitoring of engrafted cells, especially when the cells have been cultured in media supplemented with low concentrations of iron

Development and appraisal of MRI contrast agents for the in vivo analysis of stem cell grafts

The work reported in this thesis deals with the need for efficient in vivo tracking and monitoring of grafted cells and their subsequent survival. It is considered in the context of cell transplantation for neurodegenerative disorders, particularly Huntington’s and Parkinson’s disease, although the scope for a good contrast agent to monitor cells in vivo goes far beyond this. There is currently no routine method used to follow cells in vivo and it is crucial for the advancement of cell transplantation studies, both in terms of providing powerful longitudinal analysis and for decreasing the number of animals necessary per experiment. MRI allows good contrast resolution and provides details on soft tissue anatomy without being harmful to the subject. By labeling cells with an MRI contrast agent the labeled cells can be distinguished from the surroundings and information on location is attained. A good MRI contrast agent can provide more information than this though, but to date there hasn’t been an MRI contrast agent developed that can simultaneously provide good signal and be reflective of the graft changes, while not affecting the cell’s viability. The feasibility for in vivo MRI scanning of three MRI contrast agents were tested and detailed below. In Chapter 3 we looked to utilise SPIOs as contrast agents. Since SPIOs are the most widely used of contrast agents they were tested in mouse ES cells, expanded whole ganglionic eminence and rat ventral mesencephalon and successfully labeled all cell types. Problems were discovered in reference to the needle track leaving an MRI visible track that eclipsed the area of graft deposition, and while SPIOs did not hamper graft survival, only large grafts extending out from the needle track could be reliably measured. Chapter 4 examined the generation of ferritin constructs for use as contrast agents. Transgenes based on the ferritin subunits provide MRI contrast by increasing the iron content of a cell. Both subunits, heavy and light, were transfected into mouse ES cells and expressed to improve signal compared to overexpressing the ferritin heavy transgene alone, which has been done in the literature. The expected change in T2 relaxation compared to control cell lines was observed in vitro. In Chapter 5 the applicability for in vivo use of Chemical Exchange Saturation Transfer (CEST) was tested. CEST involves the selective saturation of protons of particular compounds that are then indirectly detected through the water signal. Current 2D RARE scans to pick up CEST are slow and not really transferable to animal studies, due to the large increase in scan time for each extra image slice required, here alternative 3D FLASH scans were developed that still allowed the CEST contrast change to be observed over a whole sample in a reasonable time frame. A transgene based on CEST was tested in vivo and expression was lost even under antibiotic selection. Work in this thesis contributes some understanding towards the promises and pitfalls of three MRI contrast agents, two of which may ultimately be used more routinely for cell tracking in the future