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RL1-00674-1: Derivation of new human pluripotent stem cells using small molecules

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

Pluripotent stem cells have the potential to treat a variety of human diseases, including diabetes, cardiovascular diseases, Parkinson’s diseases and Alzheimer’s diseases, in cell replacement therapies. Human embryos are the main sources for producing human pluripotent stem cells that are genetically unmodified so far. Alternative methods for producing pluripotent stem cells include somatic cell nuclear transfer or therapeutic cloning, which needs unfertilized or newly fertilized eggs, and has been experiencing both ethical and technical difficulties. Recent progress in reprogramming by retroviral transduction of a cocktail of four transcription factors offers a solution to these problems because no embryos or eggs are needed in this system. However, the use of retroviral transduction represents a serious hurdle to the use of these induced pluripotent stem cells for therapeutic application, because retroviral transduction can cause activation of cancer-inducing genes and are therefore risky. The goal of this research is to create new pluripotent stem cells that are genetically unmodified by inducing endogenous pluripotency regulators using small molecules. Screening for small molecules that induce endogenous pluripotency factors to replace retroviral delivery of reprogramming factors will allow safer production of customized patient-specific stem cells from patient’s own somatic cells and open up new avenues into stem cell therapies. It will make the dream of stem cell-based therapies come true in the foreseeable future.

Statement of Benefit to California (provided by applicant)

The establishment of pluripotent stem cell lines opened a new era in biomedical research. Human pluripotent stem cells provide great hope to be used in cell replacement therapy for a variety of human diseases. The derivation and propagation of new lines of human pluripotent stem cells from somatic sources using small molecules will allow production of patient- and disease- specific stem cells in a safe and efficient manner. These virus-free pluripotent stem cells will have important research and clinical application for understanding, diagonosing and treating serious injury and diseases that Californians may suffer. Californians will therefore benefit tremendously from the outcome of the proposed research. In addition, the development of new methods to derive genetically unmodified human pluripotent stem cells that do not require the use of either human embryos or eggs will reduce the ethical concerns of Californians for stem cell research and will stimulate other stem cell-related research in California. This research will undoubtedly bring enormous benefits for California.

Review

The applicant proposes to develop new pluripotent cell lines using high throughput screening of a small molecule library to identify compounds that induce reprogramming in human fibroblasts. S/he will employ a reporter assay to screen for the transcriptional activation of two pluripotency regulators believed to be central to embryonic stem cell (ESC) identity, and then will determine if the identified compounds can be used to generate induced pluripotent stem cells (iPSC).

The proposed work could lead to the identification of molecules useful for deriving new pluripotent cell lines from somatic cells without the need for retrovirus-mediated gene transfer, an important and significant goal. The approach constitutes a high-risk screening experiment, but it is likely, given the applicant’s expertise and environment, s/he will be able to perform this screen.

Reviewers praised the reporter construct design but differed in their assessment of the overall technical approach. One reviewer felt that the design of the screen is viable from a technical point of view, but another reviewer raised several questions with regard to feasibility. The applicant may identify molecules that are potentially useful, but in order to sort out hits from the high throughput screening campaign, and to identify those compounds that are worthy of further characterization, the expertise of a medicinal chemist will be necessary. However, an appropriate collaborator is not included in this proposal. Furthermore, the applicant prefers to identify a compound that activates both targeted genes but does not consider that such a compound may non-selectively activate a large number of genes through a broad and general mechanism. Thus, s/he needs to develop a secondary assay to rule this out. In general, follow-up assays that verify the purported activities of the compounds identified in the screen were inadequately described.

In addition to these technical concerns, reviewers questioned whether the biology of reprogramming was adequately considered in the proposed screening approach. Dedifferentiation seems to be a sequential process with multiple steps involved, and reactivation of the two targeted genes is clearly a secondary event in this process. Thus, molecules that directly regulate the two genes can help to enhance or speed up the reprogramming process but will not be the ones that truly reprogram. Furthermore, it is possible that combinations of molecules that target multiple pathways and that individually produce a weak effect are required for reprogramming, and if so, the screen as proposed may not yield any active molecules.

The following Working Group members had a conflict of interest with this application and were therefore recused from participating in review of, discussion of, and voting on the application:

• None