Funding opportunities

Derivation of hES Cells in Novel Key Regulators of Cell Survival and Growth

Funding Type: 
New Cell Lines
Grant Number: 
RL1-00661
Funds requested: 
$1 351 277
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
Human ES (hES) cells have the dual abilities to self-renew and differentiate making them powerful tools to study and treat diseases. Normal hES cells are a renewable source of differentiated cells to use in cell-based therapies. Disease-specific hES cells lines are powerful tools to study disease etiology and test disease treatments. Less than 30% of embryos give rise to hES cell lines. Given that embryos, particularly disease-specific embryos, are scarce and cells grown in the absence of key growth factors often accumulate genetic changes, improvements in the method used to make hES cell lines would be beneficial. This project will fill vital gaps in our knowledge about key signaling factors in the growth of hES cells and improve the method used to isolate and maintain hES cell lines. We previously demonstrated that neurotrophins (NTs) are potent survival factors for hES cells. Our preliminary data suggest that NT receptors are expressed in the inner cell mass (ICM) cells of the human blastocyst-stage embryo, the source of hES cells. We propose to test the ability of NTs to improve the survival of ICM cells and increase the efficiency of making new hES cell lines. The hypothesis is that NTs are important for embryo growth and will improve the efficiency of deriving new hES cell lines. The growth of hES cells depends on a balance of signals acting through multiple signaling pathways. We propose to fill the gaps in our knowledge of the pathways that control hES cell survival, proliferation, and differentiation. We established a system to perform a high-throughtput screen in hES cells to identify factors that affect hES cell survival, proliferation and differentiation. We propose to screen a high content siRNA library to identify kinases and phosphatases that affect hES cell growth. The hypothesis is that high-throughput screening can identify key regulatory pathways controlling hES cell survival, proliferation, or differentiation. The target molecules identified in Aim 2 are druggable. We propose to use those drugs (agonists or antagonists) to determine their effect on hES cell growth and stability. Genetic stability will be assayed by comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) analysis. Drugs that improve the growth and stability of hES cells will then be used to isolate new hES cell lines and the efficiency compared to that of current methods. The hypothesis is that drugs that target kinases and phosphatases identified in Aim 2 as key regulators of hES cell growth will improve the efficiency of making new hES cell lines and ensure their genetic stability. The results of this study will improve the ability to establish hES cell models of human diseases, even those for which the disease-specific embryos are scarce. Further, the improved culture conditions will reduce the probability of accumulating genetic changes that can occur when cells grow in adverse conditions.
Statement of Benefit to California: 
Stem cell biology holds tremendous promise to decipher and treat human disease. Human ES (hES) cells have the dual abilities to self-renew and differentiate making them powerful tools to study and treat diseases. Normal hES cells are a renewable source of differentiated cells to use in cell-based therapies or drug screens. Disease-specific hES cells lines are powerful tools to study disease etiology and test disease treatments. Several roadblocks remain before the full potential of hES cells can be realized. These include inefficiency in establishing new hES cell lines and their poor growth. Only about 30% of embryos give rise to hES cell lines. Given that embryos, particularly disease-specific embryos, are scarce and growth in the absence of key growth factors can contribute to genetic instability, improvements in the method used to make hES cell lines would be beneficial. The studies proposed here will fill vital gaps in our knowledge of the key factors for optimal growth of hES cells and improve our ability to isolate genetically stable hES cell lines. Improvements in the efficiency of establishing hES cell lines and their quality will allow us to establish models of a variety of human diseases, even those for which the disease-specific embryos are scarce. These models of human disease can be used to study the disease pathology and will eventually yield effective treatments. Human diseases have a detrimental effect on personal freedom and earning power of the patients and create a financial burden for their families and California. Knowledge gained from these studies could aid in developing therapies that might benefit many people.
Review Summary: 
Executive Summary The Principal investigator (PI) for this application proposes to test the ability of neurotrophins (NTs) to improve the survival of inner cell mass (ICM) cells and increase the efficiency of making new human embryonic stem (hES) cell lines. Second, a high throughput screen using inhibitory RNAs will be utilized to identify key regulatory pathways controlling hES cell survival, proliferation and differentiation. Finally, small molecules (agonists or antagonists) that target the activity of the molecules identified by the high-throughput screen will be tested for their effect on growth and genetic stability of hES cells. The rationale for this project is based on the observation that NTs are potent survival factors for hES cells and NT receptors are expressed in the ICM cells of human blastocyst-stage embryo, the source of hES cells. There was a general agreement among reviewers that the idea of improving the efficiency of hES cell derivation and their stability is a laudable goal. Reviewers agreed that the first aim may result in new hES cell lines being generated and may show an improved efficiency of derivation with NTs, though the investigators admit that statistical significance may not be reached. Regarding the second and third aims, reviewers were concerned that these are somewhat tangential to the goal of the RFA. Moreover, they are based on the assumption that specific and selective small molecules are available to target the molecules identified by high-throughput screening. Another weakness is the assumption that modulators of kinases and phosphatases, proteins targeted by the screen, are key for improving efficiency and stability of producing hES cells. In addition, reviewers felt that the resources, time and efforts that will be required to perform the high-throughput screen and subsequent identification of small molecules that target the specific proteins for their effect on growth and stability of hES cells, as envisioned in specific aims two and three, will be beyond the scope of this application, as no preliminary data is provided by the applicant relevant to these aims. Thus, overall the enthusiasm for the proposal was low. Reviewer Synopsis: Proposal based on observation that neurotrophins (NTs) are survival factors for hES cells. Aim 1 is to see if addition of NTs to the medium improves hES cell derivation. Aim 2 is to identify pathways involved in hES cell survival through siRNA screen. Aim3 is to use drugs to targets of aim 2, particularly kinases and phosphatases, to improve efficiency of establishing lines. Reviewer One Comments Significance: Increasing the efficiency of hES cell derivation is a laudable goal, as is defining factors that improve growth and viability by other methods. Feasibility: Issues: 1. The PI, an Asst Prof, is a collaborator on a comprehensive CIRM grant to assess NTs and hES cells entitled “Improve hES Cell Growth and Differentiation”. Hard to see how this isn’t the same as aim 1 (Staff note: during the review meeting, reviewers were reminded that scientific overlap will be considered during administrative review, should this application be funded, and should not be considered during the scientific review). 2. Aim 1 is reasonable but aims 2 and 3 represent entire projects by themselves that are somewhat tangential to the RFA. It is non-trivial to perform the siRNA screen described, and identifying drugs that target the targets isn’t so straightforward. 3. I find this application too all-encompassing without the preliminary data relevant to aims 2 and 3 to make a cogent case for funding. Responsiveness to RFA: Aim 1 is responsive. Other aims are tangentially related. Reviewer Two Comments Significance: The investigators have previously shown the importance of neurotrophins for hES cell survival. They will test the hypothesis that NT will improve hES cell derivation efficiency. They also propose to perform a siRNA screen to identify key pathways that affect hES cell survival. Next, they will test the pathways identified in the siRNA screen by using a kinase/phosphatase agonist/antagonist drug library. Improving derivation and culture conditions for hES cells is important for maintaining genomically stable lines and generating rare disease specific lines. Feasibility: The experience of the investigators and the facilities are more than adequate. Aim 1 will result in new hES cell lines being generated and may show an improved efficiency for derivation with neurotrophins, though the investigators admit, statistical significance may not be reached. The siRNA screen is being performed on existing lines and therefore will not generate new lines, but may improve our understanding of hES cell survival. A high-throughput imaging system will be used. This is a large project and it seems rather distinct from aim 1. Aim 3, the drug screen is meant to validate results from the siRNA screen on cells in long term culture. This aim should be a separate project that follows Aim 2, if it is successful. Responsiveness to RFA: Aim 1 will results in new pluripotent human stem cell lines and responds to the RFA. Aim 2 and 3 are addressed at stem cell biology and not so much generating new lines. Reviewer Three Comments Feasibility: All key personnel have effort with associated salary included. The PI is requesting 30%; coPI with 5%, 2 post-docs for 120% combined, 2 technical associates for 40% combined, and a graduate student for 100% effort. Aim 1 is to test the ability of neurotrophins to increase the efficiency of making new hES cell lines. Efficiency is defined in this case as the proportion of embryos that make hES cell lines and the number of ES colonies at each passage. This Aim should result in new pluripotent stem cell lines to be generated. Aim 2 will be to identify key pathways that affect hES cell survival, proliferation, and differentiation. The potential pathways will be identified by screening a siRNA library that specifically targets kinases and phosphatases. Aim 3 will be to test small molecules that interact with the targets identified in Aim 2 for their effect on growth and stability of hES cells. The weakness here is the assumption that specific and selective small molecules are available for any of the proteins identified in Aim 2. Another weakness is the assumption that modulators of kinases and phosphatases are key for improving efficiency and stability of producing hES cells. Responsiveness to RFA: This proposal is in line with the RFA in that it should generate new hES cells (Aim 1) but is more focused on improving the efficiency and stability of hES cells related to signaling pathways (Aim 2 and 3).
Conflicts: 

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