Funding opportunities

In Vivo Imaging of Human Embryonic Stem Cell Derivatives and Tumorigenicity

Funding Type: 
SEED Grant
Grant Number: 
RS1-00322
Principle Investigator: 
Institution: 
Funds requested: 
$658 123
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Human embryonic stem cells (hESCs) are one of the most fascinating subjects of interest in all of biology and medicine these days. Under certain physiologic conditions, they can be induced to become specialized cells such as brain, cardiac, liver, pancreatic, and bone marrow cells. This opens up the exciting possibility that hESCs may one day be used to treat patients with Parkinson’s disease, heart conditions, hepatitis, diabetes, and leukemia, just to name a few currently intractable diseases that affect millions of Americans alone. This field of cell-based therapies to treat human diseases is generally referred to as “regenerative medicine.” Scientists who want to study hESCs or their specialized cell derivatives typically inject them into small animal models such as mice and rodents. However, researchers currently are unable to monitor noninvasively these cells after transplant. Instead, these animals are typically sacrificed for postmortem biopsy, which precludes long-term follow-up of transplanted cells. Without the ability to follow the progress of transplanted cells over a longer period of time, important insights into hESC fates in vivo have not been forthcoming. Thus, developing a novel technology to track transplanted hESCs and their specialized cell derivatives would represent a major advancement in this field that will produce wide-ranging theoretical and practical implications. Another problem with transplantation of hESCs is the potential to cause teratomas. Teratomas are disorganized arrays of cell differentiation that appear to recapitulate many of the events involved in early embryonic development. Clearly, the teratoma formation risk is a major obstacle to future clinical application of hESCs. In this proposal, we will evaluate how teratoma forms in living subjects over time using the imaging techniques that we have developed as well as how best to prevent them in the first place. Due to the serious risks posed by teratoma formation, it is necessary to induce hESCs to become specialized cell derivatives first before transplantation for therapeutic purposes. However, this process is not efficient at present despite intense efforts searching for methods to expedite it. Our team plans to tackle this problem using the latest genomics and proteomics technology. In summary, our proposal is a targeted response to the CIRM SEED grant. It seeks to develop a novel technology (molecular imaging) that will address a critical barrier in clinical translation of hESC therapy (teratoma formation) and provide a better understanding of cardiac cell differentiation process (genomics/proteomics). Our well-established multidisciplinary team has the required training, experience, and innovation to complete the project. Overall, we are confident that our proposed studies will generate significant progress in this field, in both scientific knowledge and useful therapies.
Statement of Benefit to California: 
Stem cell based therapy holds great promise for treatment of numerous diseases. Human embryonic stem cells (hESCs), for example, can transform into brain, cardiac, pancreatic, liver, and bone marrow cells. This opens up the exciting possibility that hESCs may one day be used to treat diseases such as Parkinson’s, heart conditions, hepatitis, diabetes, and leukemia. This field of cell-based therapies to treat human diseases is generally referred to as “regenerative medicine.” However, we are still at the very early stages of understanding the capability of hESCs. There are many basic research questions that need to be addressed before we can begin clinical trials involving specialized hESC-derived cells in the future. For example, we need to understand how to drive hESC differentiation into specific pathways, to control hESC proliferation, and to monitor their cell fate after transplantation. One of the most serious problems is the potential for hESC to cause teratoma after transplantation. Teratomas are benign tumors that consist of cells from different lineages. Therefore, it is critical for scientists to understand how teratomas are formed as well as to develop techniques to monitor them noninvasively and repetitively. Our research proposal is designed to address all 3 questions: (1) how to image and monitor transplanted hESCs, (2) how teratomas are formed, and (3) how to reliably induce hESCs to become specialized derivatives such as cardiac cells. We believe that answering these questions will lead to significant advances in hESC research and novel therapies. We have assembled a multidisciplinary team of experienced investigators to attack the challenges of this project. At the same time, we will train and mentor a new generation of bright students and junior scientists in the areas of technology development and hESC biology. This ensures that an essential knowledge base will be preserved and passed on for the foreseeable future.
Review Summary: 
This proposal targets the understanding of teratoma formation from hESC by direct observation and follow up of individual cells in vivo using fluresence, bioluminescence and positron emission tomography (PET). The PI proposes three specific aims. The first is to develop a novel imaging platform for tracking hESCs in vivo. The second is to monitor the tumorigenicity profile of different hESC lines. The third specific aim is to examine key regulatory processes involved in hESC differentiation. SIGNIFICANCE AND INNOVATION: One of most important limiting factors in the possible use of hESCs in clinic which needs to be overcome is how their proliferation and range of differentiation can be fully under control. Development of non-invasive techniques to track hESC derivatives and tumorigenicity is therefore essential, and there lies the major significance of the project. The proposal is also highly innovative as it links state of the art imaging technology to the service of tracking hESCs. This will inevitably shed light in the poorly understood phenomena of teratoma formation. The innovation in this proposal lies in its systematic investigation of tumor (ie teratoma) formation post-engraftment. Although the technology to be employed is nowadays fairly standard (eg molecular imaging of cell (including hESC) trafficking is now rather widely used), the work proposed - to systematically elucidate tumor (ie teratoma) formation post-engraftment - is under-addressed in stem cell research and is therefore rather innovative. This application proposes to address the following well-defined specific aims: 1) to develop molecular imaging (fluorescence imaging, bioluminescence imaging, and PET) to track engrafted hESCs in vivo; 2) to assess the effect of cell line (WA01, WA09, HUES-2, HUES-4), engraftment site (subcutaneous, intramuscular, kidney capsule) and cell number (1, 10, 100) on teratoma formation; and 3) to elucidate by transcriptional profiling and proteomic analysis regulatory processes involved in hESC differentiation. The research plan appears conceptually and technically sound and properly designed to achieve these important objectives. Possible difficulties are not identified in any detail, however. STRENGTHS: The strength of the proposal is that it combines molecular biology and imaging, two powerful tools, to tackle the problem. Despite the complexity, the author remains focused within specific boundaries. For example, 4 cell lines will be used and not all of them to establish proof of feasibility, specific aim 3 focuses on the specialty of the PI: differentiation of hESCs toward cardiac myocyte lineage. Perhaps more importantly, the results obtained from this type of observation will also have a direct impact in our understanding of tumorigenesis. There is no more powerful approach to this problem than sophisticated quantitative four-dimensional imaging. WEAKNESSES: • The percent effort of the proposal’s professional personnel - 15% for Dr. Wu (the Principal Investigator), 1% effort of Dr. Gambhir, 5% for Dr. Connolly, and 3% for Dr. Quertermous - is marginally adequate for the scope of work proposed. • Overlap with the work proposed in the SEED Grant Proposal, # RS1-00294-1, submitted by Dr. Robert Robbins from the same institution. Dr. Robbins proposal deals specifically with differentiation of hESCs into cardiomyocytes. • Lack of depth sensitivity of in vivo bioluminescent imaging, potentially skewing the results of whole-body tracking of teratomas. • Potential difficulties acknowledged only very generally and alternative plans not described in adequate detail. DISCUSSION: Both reviewers were enthusiastic about this excellent proposal. Teratoma formation is under addressed, and this group will use the the imaging environment at Stanford in their studies, which is the highest state of the art. The non-invasive imaging techniques the PI proposes to develop are "ready for prime time". The molecular biology and imaging studies are complex, but focussed. One issue is that the work is descriptive, and the question is how much does one learn by simply watching the cells? Another issue is that using bioluminescence could skew the quantitative results for different tissues at different depths. Using PET at a uniform depth should work. A more specific technical issue that doesn't detract from the story deals with in-vivo labeling of HSV-TK. It will be difficult to convert the tracer label into an in-vivo cell number. it would be better to label ex-vivo as a calibration factor.
Conflicts: 

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