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.
The goal of this project is to develop a high resolution, high sensitivity in vivo cell tracking method. The applicants plan to accomplish this by creating a device that combines the sensitivity of magnetic resonance imaging (MRI) for anatomic localization with the cell detection sensitivity of an improved positron emission tomography (PET) reporter. The applicants will use a novel, non-viral gene delivery method to introduce a non-immunogenic PET reporter gene into various human embryonic stem cells (hESC). The applicants will then differentiate the cells into endothelial cells. Using a molecular amplification strategy to increase the PET reporter signal, they will test both their ability to detect the cells as well as their function in a rodent myocardial infarction model. The applicants will in parallel fabricate a combination high sensitivity PET, high spatial resolution, small animal MRI instrument. Finally, they will perform combined PET/MRI studies to demonstrate improved anatomic localization of the PET signal.
Reviewers agreed the lack of clinically applicable, long term tracking methods to determine the fate of transplanted cells poses a significant translational bottleneck to the development of cell therapies. If successful, the applicants' novel and innovative technology could enable sensitive, simultaneous detection of a smaller number of cells’ location and viability in vivo. This technology could have a broad and major impact on the preclinical development of multiple cell therapies. Overall, reviewers were supportive of the proposed concept. However, they were split as to whether this imaging technology would resolve the clinical bottleneck faced by cell therapy. Although some aspects of this technology are already being used in clinical studies, one reviewer expressed concern that both the proposed PET/MRI instrument and studies were limited to small animals, leaving open the question whether and how this technology can be translated to larger animals and to humans. This left the panel uncertain about the eventual clinical utility of the proposed technology, and therefore, whether the technology could fully resolve the cell-tracking bottleneck.
The presented preliminary data support feasibility of the proposed project. Reviewers appreciated convincing evidence of appropriate reporter gene function in mouse (m)ESC. The applicants have also successfully used the PET reporter element in patients and demonstrated that the reporter gene does not disrupt the function of mESC-derived endothelial cells. Prototype data suggests that PET and MRI can be integrated into a single instrument. The panel found the overall plan complex and aggressive, yet achievable, and noted extensive coordination across multiple groups would be required for implementation. The review group highlighted potential technical challenges in the transition from mESC to hESC, including generation and validation of reporter lines from four hESC lines. A discussant noted the entire application depends upon successful non-viral reporter gene integration, yet the proposal lacks alternative plans in case this fails. The review group would have appreciated a more detailed description of in vivo functional testing of the reporter cell lines. Finally, the lack of in vitro/vivo safety testing of the hESC lines with integrated reporter constructs was considered a critical omission for the program as this could affect translatability of the proposed technology to the clinic.
The principal investigator (PI) is an expert in imaging physics and has assembled an excellent multidisciplinary team with respected records in imaging stem cells with reporter genes and stem cell biology. The team is strengthened by their clinical experience with a PET reporter gene method. However, the very limited 1-2% commitment of multiple key co-investigators was noted as a potential obstacle to successful execution of this complex project. Further, reviewers felt it unlikely this large, diverse team would be able to meet weekly as planned. They noted coordination would likely rely on the communication and collaboration of postdocs from the PI and co-investigators’ labs. Facilities are state of the art and the budget appropriate.
In summary, the applicants propose to develop a technology to resolve the in vivo cell-tracking bottleneck to clinical translation. Reviewers were supportive of the technology overall and felt the applicants could likely achieve the proposed aims. However, since the proposal only described an instrument suitable for small animals and did not consider the technical and safety issues of translating the method to humans, they were left skeptical of the clinical utility of this method. Therefore the application was not recommended for funding.