Tools and Technologies I
There are two main avenues of discovery open to medical scientists to find cures or alleviate suffering in people: 1) pharmaceuticals and 2) stem cells. Both of these branches of research use the emerging technological tools of imaging, genetic engineering and nanotechnology to explore new ways to manipulate molecular and cellular processes. In this project, called "Nicola", we boldly propose to do what has yet to be done, that is to make images of individual stem cells within a living animal (i.e., a lab mouse). In order to determine the location of the cells relative to recognizable structures, we will create a "dual-modality" imager that will use MRI images for scientists to see where the single cells are located within the mouse. It should be noted that other methods such as optical (visible light), PET, MRI, etc. have shown signals from multiple (usually thousands) cells but only optical microscopy of thin slices of tissue on a glass sample slide have demonstrated single cell identification. Optical methods involving very thin fibre optics threads can "see" individual cells within a living mouse, but only to a depth of 0.3 millimeters from the end of the fibre. Users of the optical microscopy methods request a new method of see individual cells within a larger volume of the intact animal. Our proposal begins with a prototype that is designed to image one cubic centimeter, which is thousands of times the volume of the optical fibre method above. Furthermore, this is only the beginning - if our method proves to be feasible the volume can expand to ultimately have clinical utility in humans. The final goal of this project then is to visualize single stem cells within a several cubic centimeter volume of a human patient- that is, to view an organ or tissue undergoing stem cell repair. This large-field-of-view microscope would function within an MRI imaging system to provide a backdrop for the physician to know the location of the cells. The current proposal is a feasibility study that pushes the boundaries of technical capability to demonstrate, for the first time, single cell imaging within a living mouse. This is a necessary first step toward the ultimate goal of following the clinical course of action of therapeutic stem cells within human patients in the future.
Statement of Benefit to California:
Nuclear imaging is one of the last holdouts from the vacuum tube electronics age. Currently more than 95% of all nuclear imagers sold today continue to employ vacuum tube technology. [REDACTED] is the leading manufacturer of solid-state nuclear imagers for medical research applications. [REDACTED] is primarily in the field of photon-counting, using x-rays and gamma-rays to generate images in computed tomography (CT), single photon emission tomography (SPECT), and positron emission tomography (PET). [REDACED] is at the forefront of research and development in all three of these important fields. Although CT has used solid-state electronics for about 20 years now, [REDACTED] is at the forefront in R&D efforts to count and record the energy of each detected x-ray photon; all previous CT scanners have integrated thousands of photons into just one signal. The potential gains in information transfer for photon-counting CT technology are only beginning to be articulated but they are expected to be a quantum leapover conventional CT. With SPECT, [REDACTED] has introduced solid-state CZT for medical imaging research in both small animal imaging as well as clinical breast applications. In PET, [REDACTED] has introduced another solid-state detector based on avalanche photodiodes (APDs). For the current proposal, [REDACTED] has created a super-high resolution, silicon “Megapixel” detector with 50 micron intrinsic resolution. Again, this allows nuclear imaging without the use of vacuum tubes. With no vacuum tubes,the Megapixel detector is MRI-compatible and can be used for the proposed dual-modality application. The high-performance imaging technology and the systems engineering prowess of [REDACTED] are unique to this California company and superior to any that can be found in the world. The stem cell research group at [REDACTED] in regenerative medicine is led by [REDACTED]. This group has extensive experience in all modalities of imaging of stem cells – many types of optical microscopy, nuclear techniques, MR, ultrasound, and x-ray CT. They have studied the shortcomings of each modality and measured them against their needs. They desperately require an in vivo imaging modality that can identify the location of individual stem cells. The expertise in research with stem cells, labeling them, modifying them, studying their differentiation and survival in vivo – all of these reside in the world-class California-based research team at [REDACTED]. [REDACTED] and his staff have worked together with the [REDACTED] o perform preliminary experiments and to identify the need for the proposed technology. We believe this unique combination of instrumentation and stem cell expertise are a valuable natural research that only resides within the State of California.
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.
The project entitled “A novel SPECT microscopy system for 3D imaging of single stem cells in vivo” achieved significant progress toward the creation of an imaging tool that will ultimately find use in monitoring clinical stem cell therapies in humans. The imaging technique employs cutting edge technologies both in the preparation of the stem cells to be imaged and in the microscope system that we developed to visualize the stem cells in vivo. Certain radioactive atoms emit low-energy x-rays and gamma-rays when they undergo decay. The straight-line trajectories of these photons are unperturbed through a few millimeters of tissue, enabling high resolution image reconstruction when special lens-like, image-formation devices known as “coded apertures” are employed. The components of the microscope are: 1) the pixellated detector; 2) the coded aperture foil; 3) the motion control system; and 4) a radioactive calibration source. The design of each component and their relative geometrical configuration result in a system that is designed to produce cellular resolutions (on the order of 20 micrometers). The detector is the state-of-the-art TimePix detector developed as part of the international high-energy physics collaboration at CERN (Geneva, Switzerland). This detector has a special thickness of silicon that is responsive to the low-energy photon emission but relatively transparent to higher energy photons from the same radionuclide decay. The TimePix also has the necessary spatial resolution to achieve the targeted image resolution. Acceptance tests of the TimePix show that the low energy photon emission from the decay of 57Co can be detected and their energy value is discerned. The coded aperture foil has a special hole pattern called a modified uniformly redundant array (MURA) – this pattern has two properties: high acceptance of photon flux (because there are many holes), and ability to form an image of individual cells as if they are “stars in the sky” – that is, high contrast bright spots in a blank background. Our coded aperture is formed by thin layers of gold that have the hole patterns aligned to form a conical acceptance angle. For this initial aperture foil, we have selected a 30 micrometer hole size, with finer resolution planned once the performance of this prototype is understood. Precision motion control is enabled for both the coded aperture foil (for focus) and the specimen (for field-of-view). The microscope is designed to image an area of roughly 5 mm x 5 mm (variable depending upon magnification). The specimens that can be imaged range from microscope slides with radiolabeled cell populations to calibration phantoms that simulate cells of known radioactivity and location. The microscope was also designed to image radiolabeled cells injected into the spine or lower limbs of living laboratory mice. As the microscope is designed for low energies, other imaging devices are available for seeing the cells in larger volumes of interest and within their anatomical context. In particular, the translation from microscope to in vivo small animal imaging is done with a new microSPECT/MRI multi-modality instrument. The microSPECT has the ability to image the higher energy emissions from the same labeling radionuclides (e.g., 111In, 99mTc). The ability to image stem cells with hinges upon the technique used to incorporate the radioactive atoms to the cells. This project has made an exciting advance in the imaging technology needed to see the cells in vivo by incorporating “reporter genes” into the mesenchymal stem cells before they are injected into the laboratory animal. The reporter gene technique fundamentally transforms the use of nuclear imaging of cells. In conventional nuclear labeling, cells are visible only until the radioactivity that was originally attached ex vivo has decayed. With the reporter gene technique, however, we have demonstrated in laboratory animals the ability to visualize the cells in vivo in mice in three powerful and unique ways: 1) as the stem cells undergo mitosis in vivo, their progeny express the reporter imaging receptor that absorbs radioactivity injected at any time point after the genetically-engineered cells were inserted; 2) the expression of imaging receptors can be made to be promoted by differentation of the stem cells, generating an imaging signal when the stem cells have differentiated; and 3) non-radioactive fluorescent contrast agents can target the imaging receptors for optical microscopy of the stem cells and their differentiated progeny. Another feature of nuclear imaging of cells is the ability to image two photon energies – one can be from the stem cells and the other from a radiopharmaceutical that is targeted for the desired stem cell reparative function. This dual-energy imaging can monitor the location and action of stem cells in clinical trials of stem cell therapies.