Targeted delivery and MRI tracking of magnetically labelled stem cells

Stem cell therapy provides the prospect of an exciting and powerful treatment to repair or treat damaged tissue in the body. Stem cells are produced by the body as a 'universal' type of cell that is capable of replacing many tissues when they are damaged or worn out. Human stem cells are found in small numbers in tissues, such as bone marrow. These cells can leave the site at which they are made and travel via the circulation to the area of injury, thus helping to keep the tissue in good health. Stem cells can change or develop into mature cells that have different specialized functions. To put it another way, stem cells can change into different cells from their organ of origin, for example bone marrow stem cells can change into heart muscle, skeletal muscle or even brain cells. Therefore these cells can be used for organ or tissue regeneration or repair. One of the major problems with using stem cells therapeutically is that stem cells do not automatically home to the area of damage. This study will try to develop a new technique that will allow the stem cells to accumulate at the site of injury, and we hope that this will improve the therapeutic benefit of the stem cells. To do this, we have chosen to work with blood stem cells as they are know to repair damaged to the heart or blood vessels. Our previous work has shown that we can incorporate tiny iron oxide particles into the stem cells by incubating these particles and stem cells in the same dish. When these iron particles come into contact with a magnetic field they become attracted to the magnet. Therefore by placing the iron oxide particles into the cells we can magnetically tag each cell and attract these cells toward the magnet. This does not require a large magnet, and using a magnet the size of a small coin would be enough to attract these particles. To get the magnetically tagged stem cells to go the area of damage, we will place a magnet on the outside of the body over the area of damage. To cause a small area of damage we will injure a vessel wall by inflating a small balloon inside one of the major arteries in the neck (common carotid artery) of the rat. Afterwards we will inject the magnetically tagged stem cells into the circulation via a vein. From previous studies, we know that after damage to the heart only 2% of the injected stem cells will go to the site of damage. We hope to improve this number by placing the magnet on the neck of the rat, near the damaged artery wall, to attract the stem cells to this area as blood circulates through it. Next we will monitor how many stem cells have attached to the blood vessel using magnetic resonance imaging (MRI), as the magnetic iron-oxide particles appear as dark areas on the image. Finally, we will look at the vessel with different types of microscopes to see to where stem cells have attached to, and will also use an iron detector know as a SQUID to measure how many iron particles have attached to the vessel wall. This will enable us to determine if, by placing a magnet on the surface of the body, we can improve localization of the stem cells to the site of damage. We believe that if this novel technology is successful we will be able to target delivery of stem cells to other regions of the body, such as to the heart, for the restoration of function in damaged or diseased tissue. It may even be possible to use stem cells as a gene delivery system. Stem cells can be incorporated with additional new genes that have been specifically designed to fight diseases. Therapeutic genes can be introduced into stem cells before their injection, so they can transport the gene, possibly under magnetic control, to the area of the body lacking or needing a particular gene, leading to an improvement in well-being.

Great promise has been shown recently by the discovery that adult stem cells exist and can improve heart function when injected into the myocardium or the coronary arteries. However, there is limited localisation of stem cells to the myocardium after infarction following systemic injection in an animal model (approx 2%). The aim of this proposal is to label stem cells with superparamagnetic iron oxide nanoparticles, which become magnetic in the presence of a magnetic field, and to attract labelled cells to the site of injury by applying an external magnet. Superparamagnetic nanoparticles offer attractive possibilities in biomedicine. Firstly, they have controllable sizes ranging from a few nanometres up to tens of nanometres, which places them at dimensions that are smaller than or comparable to those of a cell (10-100 um), a virus (20-450 nm), a protein (5-50 nm) or a gene (2 nm wide and 10-100 nm long). This means that they can be incorporated into a cell, thereby providing a controllable means of 'tagging'. Secondly, the nanoparticles are magnetic, which means that they can be mechanically manipulated by an external magnetic field gradient. This 'action at a distance', combined with the intrinsic penetrability of magnetic fields into human tissue, opens up many applications involving the transport of magnetic nanoparticles, or of magnetically tagged biological entities. In our study, we want to use this property to enable site-specific localisation of magnetically tagged stem cells by the use of an externally applied magnetic field. Thirdly, MRI relaxation times, in particular T2*, can be shortened by the use of superparamagnetic contrast agents: several agents are now commercially available, such as ferumoxide. The superparamagnetic iron oxide particles used are magnetically saturated in the normal range of magnetic field strengths used in MRI scanners, thereby establishing a substantial locally perturbing field which leads to a marked shortening of T2*, thus offering an approach to image these particles. Several groups, including ours, have now demonstrated successful labelling of various cell types with superparamagnetic iron oxide nanoparticles, which can be imaged with MRI. Increasing evidence supports a role for endothelial progenitor cells (EPCs) and bone marrow mononuclear cells in therapeutic vasculogenesis. Tissue injury, including major burns, myocardial infarction, or coronary artery surgery, mobilises EPCs from bone marrow into peripheral blood. Animal models of ischaemia and infarction have shown that mobilised EPCs home specifically to the site of injury and differentiate into mature endothelial cells, promoting structural and functional repair. In these models, the injection of ex-vivo expanded EPCs or stem and progenitor cells has been shown to significantly improve blood flow and cardiac function and augment capillary density and neovascularization of ischaemic tissue. Initial pilot trials indicate that bone-marrow-derived or circulating blood-derived progenitor cells are useful for improving blood supply of ischaemic tissue. Furthermore, evidence for the role of EPCs in angiogenesis has been demonstrated from animal models of tumour angiogenesis. In this study we aim to magnetically label endothelial progenitor cells and mononuclear cells with superparamagnetic nanoparticles. Using in vitro cell cultures, we will assess the effects of an externally applied field on labelled cells in culture. Subsequently, we will investigate the uptake of labelled cells in a rat model of vascular damage. Varying both blood flow and magnetic field strength in vivo will enable assessment of the effects of flow rates and field strength on localisation of the label cells. If successful, this technology may open a new area of investigation for site-specific delivery of stem cells or genetically altered cells.