A Novel SPECT microscopy system for 3D imaging of single stem cells in vivo
Individual stem cells can be visualized within the body – “in vivo” by genetically engineering the cells to absorb a contrast agent when the agent is present within the body. This “reporter gene” imaging technique works not only with the originally engineered cells but also with the progeny cells that result from splitting and differentiation of the stem cells. This project involves two branches of science to achieve the goals of visualizing individual cells in vivo: 1) reporter gene engineering and testing in stem cells and their progeny; and 2) development of new imaging hardware, a gamma microscope, to visualize the individual stem cells in vivo. The reporter gene engineering effort is led by Professor Dan Gazit of Cedars-Sinai Medical Center. Radioactive labeling of cells is an established technique and our research group demonstrated the ability to image 111In labeled mesenchymal stem cells for up to 10 days. This technique is limited by the half-life of the nuclide (in this case 111In disappears with t1/2= 2.8 days), and the halving of the radioactivity when the cell divides. Cedars’ researchers successfully tested a new reporter gene method known as “human sodium iodide symporter, or hNIS)” during the first year of this project. Stem cells containing hNIS (through transfection) absorb the radioactive nuclide 99mTc preferentially – this means that hNIS stem cells (or their progeny) will absorb free 99mTc whenever it is injected into the body – days or even months after hNIS stem cells are introduced to the body. This is a dramatic improvement in the ability to visualize stem cells the hNIS gene stays with the descendents of the originally transfected cells and they will temporarily “shine” or become visible when 99mTc is injected into the body as a contrast tracer. Prof. Gazit’s research group accomplished three major goals relating to hNIS imaging of mesenchymal stem cells (MSC) during the first year: 1) transfection of MSCs with hNIS; 2) in vivo imaging of hNIS-labeled MSC in a mouse; and 3) imaging of stem cell differentiation. The imaging of stem cell differentiation is possible by linking promoters – in this case a bone-forming promoter known as human Osteocalcin (hOc) – with the expression of hNIS. This linkage of promoters associated with differentiation is done during the transfection process. Cedars’ research group successfully demonstrated the expression of the reporter hNIS in MSCs that had differentiated into bone-forming cells – demonstrating the ability to image stem cell differentiation long after the transfection of the reporter gene into the original MSC population. The research group at Gamma Medica-Ideas is responsible for developing the microscope that is capable of visualizing individual stem cells in vivo. The microscope is based on imaging low-energy gamma-ray and x-ray photons that result from the decay of radioactive labels such as 111In and 99mTc mentioned above. Unlike visible light photons, these gamma- and x-ray photons can penetrate tissue unscattered and therefore can be used in vivo for high resolution microscopy. To form the image, a type of “lens” has been developed by GM-I. This lens is a gold foil of with 100 micro-holes forming a “coded aperture” – a type of pattern that helps to identify individual cells as if they were stars in the sky. During year 1 GM-I researchers succeeded in constructing this lens which is the first of its kind- the thickest gold foil to have holes small enough to achieve cellular resolution. In order to form the coded aperture images, GM-I has selected a 55-micron pixel silicon detector of thickness 1.0 mm. This detector has the ability to record only the photons of low energy that have traversed the coded aperture holes while rejecting higher energy photons from the radioactive decays. In order to test the microscope, a miniature pattern of cell-sized reservoirs in a microfluidic slides is being developed. The challenges of correcting blurring that results from motions associated with life – pulsatile blood flow from heartbeats; breathing, tissue settling, muscular motion, GM-I is developing correction techniques that follow the motions in real time and compensate for motion blurring of the cells under study. Finally, the GM-I and Cedars-Sinai research team has developed a technology development strategy that translates the gamma microscope technology, useful for in vitro and in vivo mouse imaging, into future clinical applications involving the monitoring of stem cells and their progeny as well as the biological processes associated with the differentiation stages.