Tri-resolution Visualization System for Stem Cells and Tissue Regeneration Monitoring
The 3D imaging techniques of CT and MRI have virtually eliminated the need for exploratory surgery – a procedure which was common in difficult cases just 20-30 years ago. Not only is imaging used to discover the extent of disease, it is now used to measure the effect of therapies. The “size” of a tumor is stabilized under effective treatment – and this arrested growth can be measured with CT or MRI. New “molecular imaging” techniques (eg, SPECT) can create images of the biological processes associated with the cancer – the aggressive metabolism of cancer cells and the invasive signatures of uncontrolled growth. Images of the cessation of these processes provide a much earlier (hours-days rather than weeks-months) assessment for physicians to decide quickly upon alternative treatments if the therapy is not working.
We propose to create an imaging tool for stem cell therapies that combines the strengths of two powerful imaging modalities currently in use in both pre-clinical research and clinical practice: MRI and SPECT. Our goal is to translate this tool to the clinic to provide answers to three fundamental questions of any stem cell therapy: 1) where are the stem cells located? 2) what is the status of the stem cells? and 3) is the curative biological effect taking place? The SPECT/MRI imaging tool will be used for pre-clinical research with laboratory mice and rats. It will use MRI to provide the anatomical context – the 3D environment of the cells – by using its exquisite ability to visualize soft tissue anatomy. In the proposed pre-clinical prototype, we will use the SPECT imaging to “zoom in” on the stem cells themselves through the use of ultra-high resolution techniques that we are developing in an ongoing CIRM project. This “zoom” SPECT will be combined with the ability to simultaneously image biological processes with a second SPECT contrast agent. This use of multiple contrast agents is a unique functionality of SPECT. Our preliminary research results show SPECT imaging of both stem cells and regeneration processes in an Achilles tendon (AT) injury experiment in laboratory mice. Our unique SPECT imaging hardware is compatible with high magnetic fields of MRI. Upon the successful demonstration of the ability of MRI visualize the anatomy and SPECT to locate stem cells and to visualize the tissue regeneration in the AT model, we can begin to design the SPECT/MRI instrument for monitoring future stem cell therapies in human patients. The translation from the research lab to the human clinic is the primary component of this “tools and technologies” project. Our SPECT/MRI imager will provide non-invasive feedback to physicians employing any stem cell therapies as curative regeneration is taking place. Ultimately, a SPECT/MRI image from a scanner whose origins can be traced to this project will be the verification of a complete cure in diseases and conditions that are not being effectively treated today.
CIRM’s “Tools and Technologies” program highlights the main pathway to rapid, large-scale implementation of new ideas: the small company. [REDACTED] operate on the cutting edge – they take bold risks and create jobs, patent inventions and, as “start ups” – disrupt the status quo inertia of the larger companies. In the field of advanced medical imaging, California’s small companies and academic researchers have played the starring role in the adoption of pre-clinical imaging and the emerging field of “Molecular Breast Imaging”. In PET, which has experienced the largest growth of all imaging modalities in the past decade, practical instruments for mass production were pioneered in the 1970s and 80s. The field of microPET has also grown to have hundreds of installations in medical research labs. [REDACTED] introduced microPET designs for the small company Concorde in Tennessee, which eventually became part of the conglomerate Siemens. [REDACTED] developed a competing microPET technology in Canada which is now part of the California company [REDACTED] product portfolio.
It was [REDACTED], however, that transformed the field of small animal imaging with the introduction of “multi-modality”: SPECT/CT (2002), PET/CT (2005), and tri-modality SPECT/PET/CT (2007). These high-risk decisions in an emerging marketplace have created a standard for research on intact animal models, dramatically lowering the numbers of animals needed while improving the quality of the research. [REDACTED] is also the leader in “Molecular Breast Imaging (MBI)”, which can detect early, treatable tumors where mammography is ineffective (ie, in dense breasts). MBI can also non-invasively probe cancer biology and its response to therapies. Other California small companies, namely [REDACTED], are competing to introduce molecular imaging methods to the clinic. California has the right combination of academic prowess and business know-how to get innovative imaging technologies into the hands of researchers and clinicians. These products not only create jobs at the companies, they expand the job market in research labs and clinics where these instruments are introduced. Just as MRI has become the standard for evaluation of sports injuries, the proposed project will lead to SPECT/MRI becoming the standard for assessing the progress and success of stem cell therapies in the cure of a variety of ailments and conditions. The SPECT technology that is compatible with strong magnetic fields of MRI was first put to use in commercial products by the innovative company [REDACTED]. The SPECT/MRI development is being developed by [REDACTED] in collaboration with researchers at [REDACTED] specializing in musculoskeletal stem cell therapies.
Considerable progress has been made, despite many challenges, in the development of a stem cell microscope capable of imaging stem cells and their progeny noninvasively inside a living animal. From what we have learned from our first prototype device, we have planned significant improvements for the 2nd year of this grant. We have also developed and demonstrated a prototype device that can simultaneously acquire SPECT and MRI images of a mouse. Together with the microscope, these devices will provide tri-resolution visualization of stem cells and their tissue environment.
In parallel, we have developed and demonstrated successfully a technique to label stem cells and their progeny for observation by the microscope and SPECT. Mouse mesenchymal stem cells (MSCs) are infected by a lentivirus carrying the human sodium iodide symporter (hNIS) gene. These engineered MSCs and their progeny then overexpress hNIS which is normally found only on cell membranes in the thyroid, salivary glands, and the stomach. At any time after the MSCs are introduced into a mouse - even days or months later - they can be labeled with a radioactive tracer and imaged to track their position. An intravenous injection of Technetium-99m-sodium pertechnetate, for example, will result in the hNIS expressing cells taking up significant tracer. The stem cell microscope or small animal microSPECT scanner can then image the stem cells and their progeny.
In year 2 of this grant we will refine the imaging hardware, reconstruction software, and mouse model of Achille's tendon injury and stem cell therapy. In year 3 we will put the imaging devices and animals together to test the ability to track stem cells and monitor their tissue environment.
In year 2 the microscopic SPECT instrumentation part of the project, from the detection station (Aim 1) to coded aperture (Aim 3), has made significant progress. Ascending from the prototype, some of the rate limiting factors such as high surrounding noise and low energy specificity has been tackled which resulted in a successfully reconstructed, real image of a single cell simulating source. All critical experimental parameters, such as time of imaging, radiation dose needed, have been optimized, fully ready to move on to live imaging of real stem cell. Meanwhile, SPECT-MR instrumentation has been advanced with better collimator design and signal processing as well as with a broadened evaluation. Promising images were obtained that clearly showed non-interfered MR and SPECT of the entire mouse torso.
Stem cell establishment, after the ground breaking year 1, in which the reporting system has been effectively implanted, has gone into the stage of detailed protocol optimization. We spent a large amount of effort determining the dosing as well as the in vivo kinetics of the tracer in a variety of animals under different disease and administration conditions. Although not as shocking, year 2 is as fruitful, for detailed dynamic biodistribution profile of the tracer has been obtained and we are ready to move on to all targeted cell lines and animal models.
Year 2 the company, Gamma Medica, has suffered major organization restructuring which crippled our productivity mainly on the financial part of activities. But nevertheless we kept the research going. Now that the company has stabilized, we are ready to move into year 3, where all specific aims are to be taken to the final stage: to finalize the design and building of the prototypes and move to real life imaging: live single cell and live disease model, which will lead to final goal: commercialization of product design.
In year 3 we mainly focused on the advancement of the hardware aspect of the research, namely the main structure the microscope (Specific Aim 1), and the ancillary equipments (Specific Aims 3). We also continued on characterizing the reporter stem cells (Specific aim 2).
On the Design & fabrication of tri-resolution instrument, we continued to improve the nuclear microscope performance from several angles.
We seek to understand the tradeoffs inherent in the microscope design (sensitivity, resolution, field of view) and the effect of alignment errors by modeling the system using the industry-standard software package GEANT4 (GEometry ANd Tracking), a simulation toolkit that provides the infrastructure to visualize the detector geometry and particle interactions on the detector. We have established the Macintosh based GEANT package. We have send our researcher to software training then put to work, and produced a simple model of the collimator used in the Nuclear Microscope. We will devise a complete model of the nuclear microscope system during the extension period to inform our decisions about how to position the collimator, detector, and object.
2. Background rate and lead bricks for shielding
The nuclear microscope depends on identifying objects that are producing very low count rates so that even a small background rate from the environment can mask the signal from the object. Although selective "gates" such as an energy window is applied, some of the false signals due to background sources will still be indistinguishable from counts from the object being imaged. We purchased a set of lead bricks and built a lead housing around the system to reduce the background rate. We have managed to reduce the background so that the rate from the smallest source would still be at least 10x more than the rate from background sources. In addition, the system was placed onto an optical table (supported on air bearings) for reduction of vibration noise in the alignment of collimator and detector.
3. Large-area Sensor
We decided to establish a large-area sensor to increase the sensitivity and field of view of the nuclear microscope. A large-area collimator will be matched to the new sensor; the new collimator will have more holes than the existing collimator, and thus drive the sensitivity higher. In addition, the useful field of view of the system will be increased. After careful evaluation and testing, we have established the censoring system with a XRI QuatroSi (300 um thick silicon sensor with 2x2 array of Medipix II readout chips, 50 um pixel size, 512x512 pixel array).
4. Energy calibration
Last year we have reported hardship of the accurate and effective energy calibration, this year we improved the calibration by employing a thin indium sheets between the calibration source and sensor. This creates an input energy spectrum containing two known energies, allowing two-point energy calibration from one source.
Specific Aim 2: Stem cell experiments
In year 3 we planned to adapt microMRI with microSPECT to further characterize the stem cell activity in vivo. To better take advantage of the MR technology, we have extended our therapeutic target and established rat model where injuries were inflicted on the tendon instead of bone of the hind legs. We have implanted the cells and subjected to a few imaging experiments.
Although stem cells were proved to be active prior to implantation and tendon area showed inflammation in MRI, significant stem cell activity could not be established with SPECT. Currently we are trying to investigate different ways of the stem cell implantation as well as more flexible delivery of the Tc99m.
Specific Aim 3: Layered collimator, source, and reconstruction
1. Radioactive Phantom
The nuclear microscope has very high spatial resolution capabilities, far exceeding the ability of standard radioactive phantoms to measure. A point source phantom required to demonstrate such resolution hence has a very high precision and size criteria. Through several design and trills, we have determined on a 44 um-sized metal granules first, then make them radioactive by exposure to neutrons in a reactor, forming Cd-109. The total activity of the granules will be very small, but each granule will be radioactive and could be positioned on a glass slide as a spatial resolution and sensitivity phantom.
2. Software to extract each single gamma-ray interaction from sequence of images
We have devised and tested dedicated software programs to extract and identify single event gamma-ray interactions on the detector. Energy of event and shape of energy distribution for each event is used to partially filter out events due to cosmic rays and other background sources from the desired events from radiotracer-labeled cells. Undesired low-energy events are filtered out by use of a low-energy threshold which does not register an event for interactions that deposit energies below a set level.
In the extended period, we did our best trying to optimize the experimental condition using computer simulation. Extensive simulation with all anticipated conditions were conducted and imaging conditions such as required dose, acquisition time and energy specificity has been determined. the experiments showed that with adequate sampling of a high enough radioactivity, smaller than 200micrometer parted objects can be clearly identified. This promising results, however, required a count rate as well as specificity that real life labeling were not able to achieve. Instrumentation as well as imaging processing, therefore, need further improvement. On the other hand, Stem cell implant and imaging in vivo has found another platform: microMR, in conjunction with single photon (SPECT) scan, which could effectively identify the disposition of labeled stem cells and track in in live animals.
We are at the end of the funding period during which we have suffered numerous resource loss due to infrastructure change lead by economic downfall. We have gathered our best effort to keep the project going. The multi-aspect project has, regardless, yield positive indications that can lead development of several project, one of which has already been funded. We hold high hope that the findings in the project will lead to more meaningly commercial development in the field of molecular imaging.