Statement of Benefit to California:
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.