Creating the next generation of human iPS cells with global screens for and use of novel pluripotency factors
New Cell Lines
The recent discovery of a method to turn non-stem cells into stem cells represents a major leap forward for stem cell based regenerative medicine. These new stem cells, termed induced pluripotent stem (iPS) cells, open doors to new, patient specific stem cell therapies in the future. In theory skin cells from any patient—for example those with Alzheimer’s or Parkinson’s diseases as well as cardiac, liver or spinal cord injury—could be isolated and turned into iPS cells, then given back to the patient as a therapy. However, two major hurdles stand in our way for the use of these cells: safety and efficiency of generation. At this time, these exciting new stem cells cannot be used due to risk of the transplanted iPS cells giving the patient cancer and current methods are very inefficient as well, likely to most often fail in patients. Our goal is to solve these problems with innovative new approaches to making human iPS cells. In order to develop these methods we must identify new genes that can give the process a boost and also find substitutes for some of the currently used genes, particularly Myc, which is one of the most common genes mutated in human cancer. This one gene paradoxically is also required for strong efficiency of iPS generation. Here we propose to discover new pluripotency and self-renewal modifiers via unbiased, global screening methods. We will also tackle the complex role of Myc in iPS formation, with the objective of finding ways to replace Myc or finding a lower level of Myc that eliminates the cancer causing ability of iPS cells, while retaining or replacing its positive role in the process. If the aims of this proposal are achieved, we will have made several key contributions. We will have generated new iPS cells lines available to the stem cell field for further study with distinct and improved properties compared to existing iPS cells. We will have identified novel inducers as well as suppressors of iPS formation using unbiased global screens. The use and manipulation of these new regulators may play key roles in improved iPS protocols, paving the way for the production of yet additional new and further improved iPS lines. The goal of discovering iPS suppressors is a very innovative but logical approach. This novel strategy may pave the way to safer, non-genetic ways of efficiently inducing pluripotency using pharmacological inhibitors of these iPS cell suppressors such as drugs or growth factors. Finally, we will have also determined how to best deal with Myc’s role in iPS formation, a key step toward safe and efficient iPS methodology. Our overall goal is to produce the next generation of iPS cells that are efficient to make and safe to use. Longer term our goal is to work with our neural (Alzheimer’s disease, Parkinson’s disease, and spinal cord injury), cardiac, and liver disease teams to generate safe and effective iPS cells tailored for each patient.
Statement of Benefit to California:
The proposed studies will provide new induced pluripotent stem (iPS) cell lines and methods for generation of iPS cell lines. Such cells hold great promise for treating patients here in California, benefiting the state both in terms of improving the lives of patients as well as enhancing the knowledge of the stem cell field. It will also further the development of regenerative medicine leading to a new, valuable biotechnology. California should be a leader in developing safe, effective regenerative medicine.
Executive Summary This proposal is focused on the development of improved procedures for the generation of induced pluripotent stem (iPS) cells. The first objective is to eliminate or reduce the risk of tumor formation by iPS cells. This will be addressed by screening cDNA libraries to identify genes whose expression could replace Myc, a known oncogene, in the iPS induction process. The second goal of the project is to overcome the low efficiency of existing procedures for iPS cell derivation. RNA interference screens will identify genes whose suppression leads to pluripotent stem cell induction and support of self-renewal. In an alternative approach, cells with conditional Myc expression will be analyzed for improved induction efficiency and reduced tumorigenicity. The stated goal of improved clinical safety and efficiency in the generation of iPS cells is significant and important for the field. The approach appears innovative and likely to add new insights into the biology of pluripotent cells. However, reviewers had strong reservations about the project’s potential to improve iPS procedures and yield clinically useful cell lines. A particular concern was the use of integrating vectors which will create a problem for the subsequent clinical application of the derived cells. In addition, some of the suggested candidate genes selected to be targeted for suppression in the proposed studies are known to be tumor suppressors, thus the goal for reduced tumorigenicity of resultant cell lines may not be realized. The PI is a young researcher with expertise with the Myc gene and decent productivity. The institutional environment and facilities appeared to be excellent. However, the project’s feasibility appears limited. Defined methods for evaluating the pluripotency of the derived cell lines were not presented, and criteria for iPS cell colony selection were not addressed. This was considered to be particularly problematic because the PI has no previous experience in the generation of iPS cells and reviewers stated that the addition of a robust collaboration with an iPS cell expert would have made this a stronger application. In conclusion, although its goal is to improve procedures for the generation of clinically safe pluripotent cell lines, the experimental design would likely yield cells of questionable value for clinical use. It is unclear that the proposed research would make a major contribution in moving iPS cell generation toward the clinic. Reviewer Synopsis The goal of this proposal is to develop improved procedures for the derivation of induced pluripotent stem (iPS) cells. The proposed investigation aims at two difficulties that stand in the way of using iPS for patient-specific stem cell therapies. The first is the high risk of tumor formation, and the second is the lack of a methodology for consistent iPS generation. To address these difficulties, the PI proposes the identification of new pluripotency and self-renewal modifiers through screens, and investigation of the role of Myc in iPS cell formation. Reviewer One Comments Significance: Significance and Innovation: Innovation is very high. The applicant suggests that one can “fish” for new pluripotency factors using a complementation assay where cells transfected with 2 key iPS genes such as Sox2 and Oct3/4, would be used to search for complementation partners from a global cDNA library. The goal is to improve the safety and efficiency of iPS generation, although it was not clear how this would be measured. The second aim would search for suppressors of iPS (suPS) generation by the introduction of a lentiviral shRNA library into already established iPS dermal fibroblasts. In additional steps candidate suPS would be introduced singly or in a cocktail. The applicant suggests that inhibiting suPS might lead to more efficient generation of iPS lines. Some of the candidate genes selected to be targeted for suppression in the proposed studies includes P53, Rb and GSK3B. Given that absence or mutations in these genes can also lead to cancer development, the goal for reduced tumorigenicity of resultant cell lines may not be realized. Feasibility: Design and Feasibility of the Research Plan: The author’s lab and collaborators have experience culturing hES cells. Although they do not appear to have produced iPS lines yet, the technologies they demonstrate in other areas suggest that it will not be a problem. A defined method for investigating the pluripotency of the cell lines created by this technology is not presented, only a general approach is suggested. It is not clear how the author would select iPS colonies for expansion. Since the more experienced labs seem to do this by morphology, the lack of experience by this group in these areas would slow progress. The PI is an expert on the MYC gene and many of the experiments presented here are designed to find complimentary genes which could substitute for cMYC in iPS generation. They suggest that by replacing MYC they will reduce tumorigenicity of the resultant lines. The author suggests that the role of the cMYC gene is unclear in iPS generation because even when the gene is left out of the cocktail for transfection, the endogenous MYC genes can be expressed. Therefore he proposes to knock down endogenous MYC with tet inducible shRNA. He claims the titratable cMYC system will improve efficiency and reduce tumorigenicity. Both cMYC and N-MYC would be targeted in these experiments. The author has strong preliminary data to indicate that he can produce the MYC regulated lines. The application describes a basic science research proposal in the manner of a NIH R01 application. There is no mention that the author has considered the possibility of producing cell lines which could be used for regenerative medicine. All of the technology involves integrating vectors which could pose a problem for future clinical application. It is unlikely that any line created in the proposed studies could be used in such a manner. While the science proposed is thrilling it is not clear that the goals of the program would be met by this application. PI and Personnel: The PI, Paul Knoepfler received his Ph.D in 1998 at UCSD in molecular pathology. He is currently an assistant professor at UC Davis. He has an excellent publication and funding record for his level. His collaborators on this application are also highly qualified, however all within the basic science areas. There does not seem to be a clinical component to the application or clinical input to the planning. Except for target gene discovery, the goal for the research to move to clinical treatments will not be realized by the proposed studies. Responsiveness to RFA: Adequate Reviewer Two Comments Significance: There are two specific aims. The first is the generation of novel human iPS cell lines with reduced turmorigenicity using a cDNA library screen for new inducers of pluripotency and self-renewal. The second aim is the generation of new human iPS cell lines using shRNA screens for suppressors of pluripotency and self-renewal (suPS). The applicant’s goal of safety and efficiency in iPS generation represents a clear priority in the field, and thus enjoy a high significance. The approach in searching for new pluripotency and self-renewal modifiers is highly innovative. However, given that all iPS generation in this proposal will involve integrating vectors, it is difficult to see how they will be useful in regenerative medicine. There seems to be lack of connection between the innovative basic science of this proposal and the stated goals of moving iPS generation to clinic. Feasibility: Dr. Knoepfler is a young investigator of some (but not outstanding) productivity. There will be a collaboration with Dr. Ronald Li (1% —UCD). A more robust collaboration with other researchers in the field would make this a stronger application. In particular, a collaborator with experience in iPS generation would be a welcome addition. The PI has no previous experience in generating iPS. Vectors used are still a problem. The facilities at UCD are excellent, and will be suitable for the goals of this proposal. Preliminary data for these studies are good, suggesting that the authors will be able to generate a Myc-regulated line. Responsiveness to RFA: This proposal is likely to generate hESC lines that have the ability to differentiate into all three germ layers. The plan for sharing of new cell lines by depositing in the CIRM-sponsored stem cell bank is adequate.