Tools and Technologies II
The overall goal of the proposed research is to develop tools to accelerate the translation of highly pluripotent human stem cell technologies to the clinic, including both induced pluripotent and human embryonic stem cells (hIPSC; hESC). To this end, the proposed studies will address the most serious translational bottleneck in the regenerative medicine field: safety. The critical safety concern is the potential of therapies to form a type of tumor called teratoma. Both academic and industry researchers must now rely upon a imperfect tool developed three decades ago to address these concerns: slow, expensive, and imprecise in vivo teratoma assays. Stem cell researchers also have few tools to enhance stem cell safety as there are no known teratoma inhibitors. The lack of optimal tools to measure and enhance the safety of stem cell therapies is a critical bottleneck for regenerative medicine. Our solution to this problem is a new technology called in vitro teratoma assays. This “teratoma in a dish” technology consists of rapid, reproducible, in vitro teratoma assays that are 5,000 times less expensive per sample than the in vivo assay. We will use the in vitro teratoma assays not only to measure the safety of cells lines, but also to screen chemical libraries to discover a second type of powerful technology that enhances safety: teratoma inhibitors. In addition to measuring and enhancing safety, the in vitro teratoma assay technology can also be used to screen cell lines for pluripotency to identify bona fide hIPSC and hESC lines. In vivo teratoma assay technology is a major weakness in the field. Its methodology has remained mostly unchanged for decades relying upon injecting stem cells under the skin of immunocompromised mice. As a result, testing large numbers of potential clinically valuable stem cell lines for safety is not feasible as it could require thousands of mice. In contrast, the in vitro teratoma technology would use less than a dozen plastic multi-well plates to achieve the same goal. Our “teratoma in a dish” technology has extremely broad clinical relevance, as any disease potentially treatable by regenerative medicine must first be safety tested. The proposed research will also produce technology to enhance safety. We will use our in vitro teratoma assay system to screen for drugs that inhibit teratoma formation. These small molecules will serve as powerful tools to catalyze the advancement of pre-clinical studies to Phase I Trials. To date, the only safety measures available in the field are differentiation and cell sorting, but these are inadequate. We predict this new technology will lead to many new collaborative regenerative medicine research efforts as well. When these studies are completed we will have produced powerful new tools for stem cell researchers to both measure and enhance stem cell safety, making the proposed studies transformative for regenerative medicine.
Statement of Benefit to California:
A major bottleneck in translating stem cell research into the clinic is the issue of safety. The two most promising types of stem cells for regenerative medicine, hESC and human iPS cells, both have the ability to cause teratoma and potentially other tumors. At present, the only method to assay the tumorigenicity of these cells involves slow and expensive murine model systems, so-called "in vivo teratoma assays". The proposed research will benefit California by producing a new technology whereby the tumorigenicity and hence safety of stem cell lines can be evaluated in a rapid, affordable manner using in vitro teratoma assays or "teratoma in a dish". This new technology will save a large amount of funds for California researchers working on regenerative medicine in the form of labor, supply, and animal care costs now associated with the in vivo teratoma assays. Per sample, our in vitro assays are 5,000 times cheaper than the traditional in vivo assays. A second benefit of the proposed research is that it will identify drugs that can be used to enhance the safety of stem cells prior to their use in transplantation therapies. Currently there are no known potent inhibitors of teratoma formation. Identification of drugs that block teratoma formation will make clinical trials progress more rapidly and reduce costs, a great benefit to California. By enhancing safety, the drugs will also make regenerative medicine therapies more efficient and effective.
This proposal is focused on the development of an in vitro “teratoma-in-a-dish” assay to assess the potential tumorigenicity of human embryonic and induced pluripotent stem cells (hESCs and iPSCs). Current methods rely on in vivo teratoma assays in immunocompromised mice and are both time-consuming and expensive. The applicant identifies the lack of efficient, low-cost methods to assess the teratoma-forming potential of hESCs and iPSCs as a bottleneck to the translation of stem cell based therapies. There are three specific aims: (1) to develop a sensitive in vitro teratoma assay compatible with high-throughput screening; (2) to use this assay to test 24 candidate teratoma inhibitors; and (3) to conduct ultra-high throughput screening on commercially available chemical libraries for additional candidate teratoma inhibitors. The reviewers agreed that this proposal addresses a significant translational bottleneck and, if successful, would result in a useful tool for the field. However, they questioned the scientific rationale for studying teratoma formation in vitro. It wasn’t clear to reviewers that the growth and differentiation of pluripotent cells in a soft matrix can model teratoma formation in a complex in vivo setting. They were unsure what the in vitro assay would actually assess and what it would correlate with in vivo. Reviewers noted that while the idea of in vitro teratoma inhibitor screening is novel, embryoid bodies in culture are already known to recapitulate many of the differentiation patterns generated by teratomas. It was not clear to reviewers that the proposed approach would differ significantly from existing assays for embryoid body differentiation. Reviewers raised a number of concerns about the research plan. They noted an absence of data supporting the relevance of the in vitro teratoma model, such as morphological comparisons between teratomas generated by pluripotent cells in vitro and in vivo. The reviewers described the preliminary data as largely qualitative. They noted that the presented images were of low quality with a low signal-to-noise ratio and that these were not accompanied by statistically analyzed data. Reviewers felt that the 2D image analysis used was insufficiently accurate and cited the availability of more sophisticated software and cell tracking tools. Reviewers were also unconvinced that the proposed 3D monitoring would be feasible with the equipment described. As a technological improvement, they recommended the development of machine learning software to more accurately measure specific, defined aspects of teratoma formation, as opposed to only focusing on colony growth. Reviewers also questioned the applicant’s portrayal of the proposed drug screen as “ultra high-throughput” describing it instead as a low-throughput, potentially high content screen. The reviewers noted that the Principal Investigator (PI) is a young, successful scientist with excellent expertise in stem cell biology. However, they were concerned that the PI is the only senior investigator committing effort toward the project. There are several letters of support from other senior investigators but they lack adequate detail. These letters did not assure reviewers that there would be a sufficient level of involvement from experts in the areas of high-throughput screening and the pathological assessment of teratomas. Overall, while reviewers appreciated the significance of this proposal, and the potential impact that an efficient, low-cost teratoma assay would have on the field, they raised a number of concerns with the scientific rationale and research plan. Most importantly, they were not convinced that the in vitro teratoma assay proposed would provide an accurate model of in vivo teratomas.