Human embryonic stem cells (hESCs) are pluripotent cells derived from the inner cell mass of blastocyst-stage embryos. Their importance to modern biology and regenerative medicine derives from two unique characteristics that distinguish them from all other organ-specific stem cells identified so far. First, hESCs can be maintained and expanded as pure populations of undifferentiated cells for extended periods of time in culture. Second, hESCs can differentiate into every cell type in the body, including neuronal, cardiac, hepatic, and endothelial cells. However, a risk of using hESCs is the possibility of cellular misbehavior (i.e., teratoma formation). In addition, accurate methods to non-invasively measure efficacy of hESC therapy in regenerating function are needed at early, intermediate, and late stages subsequent to treatment. Thus, it is essential to understand the biological process of hESC differentiation in vitro and in vivo if the clinical potential of these cells is to be realized.
To date, the majority of studies on hESC fate have relied on ex vivo analysis such as histologic staining for green fluorescence protein (GFP) or beta-galactosidase (lacZ). To understand cell fate in vivo, noninvasvie techniques must be developed. This CIRM Tools and Technologies proposal seeks to develop novel biological tools to track the fate of transplanted hESC derivatives. Our multi-disciplinary team consists of experts in stem cell biology, molecular imaging, genetics, physics, engineering, and pathology. Our goal is to increase the detection threshold of transplanted stem cells by at least 10-fold using a robust non-immunogenic reporter gene, a molecular amplification strategy, and advances in PET/MRI instrumentation. Successful execution of the proposal will enable safe and controlled clinical translation of hESC-based therapies in the future. Importantly, the same platform can also be used to image the fate of transplanted adult human stem cells and induced pluripotent stem cells, both during early stages post-treatment (using the PRG signal) as well as in late stages for monitoring endpoint efficacy of cell transplantation in restoring function by measuring positive perfusion changes using a PET myocardial perfusion tracer such as 13N-ammonia, and a perfusion-weighted MRI pulse sequence.
Statement of Benefit to California:
Human embryonic stem cells (hESCs) can differentiate into every somatic cell type of the human body and possess the capacity of unlimited replication. As a result, these cells have been regarded as a leading candidate for a source of donor cells in regenerative medicine. We believe it is important to understand the in vivo behavior of hESCs and their derivatives using novel imaging technologies. This is further highlighted by recent FDA approval for Geron Corporation to start its first hESC-based therapy for patients with acute spinal cord injury.
Conventional histology and reporter genes such as green fluorescent protein (GFP) and beta-galactosidase (LacZ) do not allow for longitudinal imaging of cells because these methods require animal sacrifice and only provide a "snapshot" of the biological fate of transplanted cells. Recent advances in the field of molecular imaging have made it possible to non-invasively track transplanted cells over time. Here we seek to develop novel strategies that will enable us to track the long-term fate of transplanted hESC derivatives using a safe, non-immunogenic positron emission tomography (PET) reporter gene. The reporter gene approach that our team proposes would allow us to address challenges such as: (1) reliable tracking of stem cell viability, (2) ability to visualize long-term fate, (3) avoiding risk for insertional mutagenesis, and (4) boosting PRG expression to visualize lower abundance cells.
In parallel, we propose to develop a magnetic resonance imaging (MRI)-compatible PET insert that has 10-fold greater PET reporter gene signal detection sensitivity. This would enable visualization and quantification of 10-fold fewer cells that have been transplanted into animals, even in the presence of background accumulation in other cells throughout the body. The exact location of where cells engraft can be determined by magnetic resonance imaging (MRI). The combined, concurrent PET/MRI imaging facilitates accurate tracking and quantification of the biodistribution and survival of smaller clusters of cells, enabling early monitoring of the efficacy of transplanted cells in restoring function. Furthermore, with a PET perfusion tracer such as 13N-ammonia, and a perfusion-weighted MRI pulse sequences, intermediate and long-term efficacy of cell transplantation in restoring function can be assessed by measuring positive perfusion changes.
In summary, we believe the incorporation of our reporter gene technology and the novel PET/MRI platform will lead to significant advances in detection of low abundant cells in the field of regenerative medicine. The proposed tools can be used to study various cell types, including hESCs, human induced pluripotent stem cells, and human adult progenitor cells. Thus, the advancement made here will be highly valuable and of tremendous interest to both scientists and clinicians alike working together to develop safer stem cell based therapies.