Imaging and Tracking of Stem Cells In Vivo with Magnetic Particle Imaging

Funding Type: 
SEED Grant
Grant Number: 
RS1-00174
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
We aim to develop, test and validate a new, sensitive and affordable scanner for tracking the location of injected cells in humans and animals. This new scanning method, called Magnetic Particle Imaging, will ultimately be used to track the location and viability of stem cells within the human body. It could solve one of the greatest obstacles to human hESC therapy---the ability to track stem cells and see if the cells are thriving and becoming a cell that can improve function of damaged organs. None of the current methods to track stem cells will be useful for tracking stem cells through a living human. MRI is too insensitive and expensive. While optical imaging methods (fluorescence and luminescence) are useful for cell studies under a microscope, they all fail deeper than about 1 cm. Nuclear imaging methods involve radiation and offer poor resolution. Ultrasound has many obstructions and the gas bubble stem cell tags do not persist very long. Hence, we wish to develop a new imaging method tailored for tracking stem cells in the human body---Magnetic Particle Imaging. Magnetic Particle Imaging has 200x better sensitivity compared to MRI and it will be significantly (50x) less expensive; and will require no expert operator. Only developed in the last year, Magnetic Particle Imaging scanners are not available commercially. Our expected resolution is 100 um with scan times of seconds per imaging slice. Initial in-vitro tests show promise that single cell detection is quite feasible. The method employs FDA approved superparamagnetic nanoparticles (e.g., Resovist or Ferumoxtran) for Magnetic Particle Imaging. The specific aims are to (1) construct a Magnetic Particle Scanner for mice; (2) test the MPI scanner against histology; and (3) validate the MPI scanner against MRI in a cardiac infarct mouse model. An inexpensive ($40,000) high-resolution, simple stem cell scanner is absolutely critical for the field of stem cell therapy to progress to humans. Research on mESC is funded heavily by the NIH, but this research is motivated principally to track hESCs in humans and, hence, is very unlikely to be funded by the Federal Government.
Statement of Benefit to California: 
Stem cell therapy has enormous promise to become a viable therapy for a range of illnesses, including cardiac disease, diabetes, stroke, and alzheimer's. If we could expedite the development of these therapies, it would be of enormous benefit to both the citizens of the State of California, since they and their relatives would enjoy far less disability. Moreover it would greatly reduce the Medicaid costs for the State. The diseases mentioned above are the leading cost illnesses as measured in lost productivity, lost wages, and extended care of the disabled. A study of the 1987 National Medicaid Expenditure Survey and the 2000 Medical Expenditure Panel Survey showed the 15 most costly medical conditions are (1) heart disease ( 8%), (4) cancer (5%); (5) hypertension (4%); (7) cerebrovascular disease ( 3.5%) (9) diabetes (2.5%). A key obstacle to stem cell therapy is the inability to track stem cells through a human body. This means that there is no way (other than measuring organ function) to determine how well the therapy works. Considering the number of delivery methods and the number of challenges to getting stem cells in place, and then coaxing them to differentiate and improve organ function, it will be impossible to optimize the entire process without intermediate imaging feedback to optimize each step. Unfortunately there is no acceptable method now for tracking stem cells throughout the human body. The new method, called Magnetic Particle Imaging, to be developed in this research does offer a way to track stem cells. Moreover, it will be inexpensive and quite simple to operate. The research requires a collaboration between imaging bioengineers, stem cell biologists, and cardiologists. Fortunately, we have been able to form such a team between Berkeley and Stanford. We also have formed a key collaboration with Dr. Nick Van Bruggen of the Bioimaging Group at Genentech Corporation, which is very interested in this research for their own business. Hence, we are very excited to begin this research so the basic technology will be in place once the complex biology of stem cells is worked out.
Progress Report: 
  • Full clinical potential of human ES (hES) cell therapy can be achieved when one can grow hES cells effectively while maintaining full pluripotency. We have focused on developing stem cell culture media by which we can maintain pluripotency of human ES (hES) cells. It is critical to determine and develop a chemically defined media that are animal product-free and feeder cell-free conditions so that the media can be standardized throughout stem cell research and in clinical situations.
  • One major recombinant protein component we will use in developing chemically-defined media is a set of TGF-beta signaling ligands, receptor domains, and ligand-specific antagonists. We have established a new method of generating a diverse array of these ligands, including BMPs, Activins, inhibin, and their heteromeric ligands of the BMP/Activin class ligands. Some of these heteromeric ligands possess their signaling properties unlike their homodimeric counterparts. These reagents include Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, and their derivatives. These reagents have been engineered by chimeric recombination. They were also further modified by site-specific mutagenesis, and by combinatorial heterodimeric assembly to create and modify protein-specific binding affinity to their binding counterparts. Several of these reagents are now available as recombinant protein in sufficient quantity for large-scale screening for media composition.
  • To establish the functional characteristics and optimal culture combinations using these new reagents, we have used an established hES cell, H9. We have cultured H9 cells in various compositions of culture media containing some of the engineered reagent and followed expression of several differentiation markers to monitor for pluripotency of hES cells, and also for their differentiation-guiding and pluripotency-maintaining abilities. We have first examined effect of aforementioned reagents: Noggin, BMP2, BMP3, BMP6, GDF6, BMP2/6 heterodimer, BMP3 S28A mutant, in our standard culture media mTeSR condition, which does contain bFGF, for proliferation and differentiation of hES cells. In these assays, hES cell line H9 was cultured and reagents were added at varying concentration (1-100 ng per ml) over 1-5 days culture period. Reagents were added in new media during the course of cell culture. We have used morphological change and the presence of markers as a means to follow the differentiation. Ectoderm markers are Nestin, Cdx2; Mesoderm by Brachyury, HBZ; Endoderm markers by CXCR4, Sox17, Gata4, HBF4 alpha, Gata6, AFP. Two BMPs had pronounced effects in inducing cells to endoderm. We have followed up by analyzing the efficiency using FACS. Up to 60% of cells have undergone to endoderm-marked cells. With the availability of a cell sorter, we evaluated pluripotency by means of proliferation rate, morphology, fluorescent signal in the reporter lines by visual inspection and FACS, then we further characterized the factors by real-time PCR for stem cell markers and karyotyping.
  • It is known that high concentration of FGF can suppress the action of BMPs, so we planned to repeat the experiments in mTeSR media with lowered levels of FGF to re-evaluate the effects of BMPs on cell differentiation abilities. After these tests were completed, we established a protocol performing these assays in high-throughput manner. We are currently in the process of writing this work for publication (Valera et al., in preparation).
  • Towards the development of chemically defined culture media to maintain pluripotency, we have then tested various newly-engineered reagent to replace a protein component in TeSR media. We have established a combination of protein factors known to maintain established hES cells without using nonhuman products except human albumin, which include basic fibroblast growth factors (bFGF), and a bone morphogenetic protein derivative known as AB2008. We have termed this new media as CAV media. We are currently in the process of writing this work for publication (Valera et al., in preparation).

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