Funding opportunities

Derivation of new human pluripotent stem cells using small molecules

Funding Type: 
New Cell Lines
Grant Number: 
Funds requested: 
$1 324 404
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
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: 
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 Summary: 
Executive Summary 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. Reviewer Synopsis The goal of this proposal is to identify small molecules that can activate the endogenous Oct4 and Nanog locus in human cells. Proposed is a screen based on a dual luciferase high-throughput reporter, then to test if the identified compounds can be used to produce iPS cells and finally to show that these new iPS cells are truly pluripotent using standard pluripotency tests. City of Hope has obtained a chemical library of 70,000 compounds and the applicant is confident that there will be components that can directly reactivate two key pluripotency genes. Reviewer One Comments Significance: Dr. Yanhong Shi proposes to develop a new pluripotent cell line using high throughput screening and a reporter assay to identify compounds that induce reprogramming in human fibroblasts. The significance is that this work could lead to the identification of molecules useful for deriving new pluripotent cell lines from somatic cells without need for virus transfection. Feasibility: The proposal asks for support for 10% of the PI (no salary, however), four co-PIs (no salary), and three postdocs for 100% effort and salary. Aim 1 is to identify small molecules that activate endogenous human ES cell pluripotency regulators Oct4 and Nanog, two transcription factors believed to be central to stem cell identity. They will use an Oct4 and Nanog promoter-driven luciferase reporter gene system. Aim 2 involves the use of molecules identified from Aim 1 for secondary assays to confirm that they promote human embryonic stem cell morphology. Aim 3 is further characterization of the derived lines including gene expression and epigenetic profiles and pluripotency potential. My major criticism is that the assumption is that compounds identified through the high throughput screen are going to be adequate for use for purposes of somatic cell reprogramming. There may be molecules that are potentially useful but they will have a number of compounds they will need to sort through to determine which are worthy of further characterization. None of the people on this proposal have a medicinal chemistry background and this is absolutely necessary to help sort out hits from the high throughput screening campaign. The cell line for the high throughput screen has not been constructed yet, and this will take three months at the minimum, if all goes smoothly. The dual luciferase construct is a good design so that they can distinguish between compounds that increase expression of either gene or both in the same cells. They need to try to choose a cell line that expresses both to a similar degree so that the same conditions can be used for both signals. A stable cell line will be much preferred, although they mention the need for transients if expression levels are too low. This strategy really should be a last resort. They prefer a compound that activates both genes but need to be careful because these compounds may non-selectively activate a large number of genes through a broad and general mechanism. They need to develop the secondary assay to rule this out. They are choosing a human fibroblast cell line that has been shown previously to successfully produce pluripotent cells. They have a 70,000 compound library that has been used productively for other screens. They mention a million compound library of natural products through a collaboration with Strathclyde Institute for Drug Research that they could use if no hits are found. Another potential weakness is that if more than one target or pathway has to be modulated to produce the reprogramming, they will not be able to identify any active molecules. Combinations of molecules that produce a weak effect are a possibility and identification of the mechanism of how the molecules may be working will be important for interpretation of the effects. They propose to use microarray data to help determine mechanism and whether any deleterious effects such as oncogenesis are noted. The PI has experience with these types of assays and high throughput screening, the co-PIs have complementary skills that will be necessary for the project. They would benefit from a medicinal chemistry consultant to help sort out the molecules they identify in the screen. Responsiveness to RFA: The proposed work fits well in the RFA for development of new stem cells using an alternative method than from embryos from IVF. The proposed work will also potentially contribute to our knowledge about reprogramming. Reviewer Two Comments Significance: This Proposal addresses a significant problem in the field, since current technologies use integrating retroviral vectors for gene delivery. Feasibility: - design is clearly feasible from a technical point of view, it is likely that given the PI’s expertise and the given environment they will be able to do this screen - problem with the biology: dedifferentiation seems to be a sequential process with multiple steps involved, reactivation of Oct4 and Nanog are clearly secondary events thus molecules that directly regulate Oct4 or Nanog can help to enhance or speed up the process but will be not the one that truly reprogram - overall high-risk screening experiment

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