Funding opportunities

High Throughput for Small Molecule Probes of hESC Pluriopotency

Funding Type: 
SEED Grant
Grant Number: 
Funds requested: 
$750 114
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
The key advantage of human embryonic stem cells (hESCs) is that they are pluripotent, meaning that they can be induced to develop into many if not all of the tissues in the body. This property makes them ideal for research into methods of tissue development and regeneration after injury, and will be key for efficient growth of the large quantities of cells that will be needed for broad patient access to forthcoming stem cell therapies. However, hESC culture requires laborious maintenance procedures to avoid spontaneous differentiation into fibroblast-like cells that exhibit loss of this key property of pluripotency. Culture additives, such as bFGF, are currently used to maintain pluripotency, but are only partially effective; thus, there is a substantial need for increased understanding of the pathways that maintain the stem cell state. The goal of this CIRM SEED application is to develop and implement a screen to identify novel small molecule probes of the pathways that maintain hESC pluripotency. The newly discovered probes themselves, or the new cellular pathway information, will lead to more efficient stem cell growth for research into treatments and for implementation of those treatments once they become available. {REDACTED} has made substantial infrastructure and personnel investment in high throughput screening of drug-like molecules for biological activities, including identification of molecules that promote stem cell functions such as pluripotency and directed differentiation into clinically important tissues. Prior studies in our lab have shown that it is feasible to screen large drug libraries for compounds that maintain pluripotency in hESCs. This proposal is to carry out a screen and to chemically optimize the hits so that they can be used as reagents in hESC culture and as probes to identify the genes and proteins in an hESC that confer pluripotency. The algorithms and procedures that constitute the screening platform and all information about the primary and optimized small molecules will be deposited publicly on PubChem ( for open access to all. CIRM funding is critical because of the need to give highest priority to follow up on hits that function across multiple hESC lines, including non-NIH registry lines. Most importantly, our screens are robotically automated and CIRM funding allows us to employ the non-NIH lines that have the demonstrated ability to be dispersed to single cells without adversely affecting their performance, a property that makes them amenable to robotic liquid handling.
Statement of Benefit to California: 
This proposal is a multidisciplinary collaboration among stem cell biologists, chemists, and engineers to address a critical problem that limits the widespread use of hESCs in biotech and eventually clinical settings. The importance of the problem and the multidisciplinary approach will lead to benefits for California that include: 1. The results promise insight into the nature of stemness, the essential property of hESCs that permit them to be cultured indefinitely yet retain the ability to form many useful tissues. The insight gained is believed to be required for understanding how to enable large scale replication of stem cells for forthcoming regenerative medicine therapies involving stem cells. 2. Bringing together a diverse set of people (chemists, cell biologists, engineers) to better address a stem cell problem and forge new links in the academic community that will be capable of creating new approaches to stem cell research. These new areas of research will be one of the legacies of the stem cell initiative and they promise to invigorate academic research in other areas as well. 3. The technologies we will develop and implement apply the new discipline of chemical biology to stem cell biology, and the merger promises to spin off new areas of investigation and biotechnology products expected to benefit the practice of medicine and the local economy. 4. Lastly, supporting implementation of the leading edge technologies and collaborations that will contribute key new components to California’s infrastructure, particularly high throughput chemical library screening and hESC culture techniques, that can be brought to bear on other areas of biomedical research, both in stem cell and non-stem cell sciences.
Review Summary: 
SYNOPSIS: In this proposal the PI will develop automated methodologies to perform chemical genomics screens to identify small molecules that promote hESC pluripotency. The first Aim will optimize the assay systems using non-NIH registry hES cell lines. The rationale is that these cells are more amenable to single cell manipulations that are required to perform these types of screens. An emphasis will be placed on developing robotic and automated assay systems. In the second Aim, a library of 50,000 small molecule compounds, consisting of known drugs and natural products that are structurally will be used to screen for molecules with effects on pluripotency. The read-out will be automated cell-based imaging analysis of nuclear Oct4 content. Primary hits will be confirmed by analysis of other pluripotency markers (i.e. Nanog) and testing whether the hES cells retain functional differentiation abilities in vitro. In the final Aim the identified hits will be further analyzed with respect to protein and signaling pathway targets. The PI will also perform cheminformatic studies to optimize the structure/activity profiles of the identified hits. SIGNIFICANCE AND INNOVATION: The mechanisms that maintain pluripotency in hES cells are poorly understood, and maintaining hESC pluripotency is a desirable outcome. There is a need for standardized methods to maintain these cells and their pluripotency in culture. Chemical genomics screens have been very useful in a number of contexts. In fact, a small molecule that allows for the maintenance of pluripotency in murine ES cells has recently been identified. While the degree of innovation is not great, the kinds of studies proposed are ideal for the questions posed and these studies have the potential to generate valuable information. Success of these studies would have broad impact, and therefore, would be of high significance. STRENGHTS: The strength of this proposal is the application of state-of-the-art chemical genomics technologies to the area of hES cell biology. The PI and collaborators are in a unique environment where such studies can be performed; San Diego is one of the few that can support studies of this scale. The three Aims are logically developed and described. These begin with extensive optimizations, which are supported by preliminary data; thus, there is reason to believe that the optimization studies would be successful. The second and third aims are also strong. The justification for all of these approaches is solid, and the use of non-NIH registry hES cells is well justified. WEAKNESSES: The major flaw of the studies proposed is that they are overly ambitious. Over a 2 year interval, perhaps the first 2 Aims could be accomplished, but only with a good degree of serendipity. It is quite likely that the studies in the third Aim are premature, as they clearly depend on the success of the first 2 Aims. More importantly, despite the environment in San Diego, this lab in particular does not have the demonstrated productivity in terms of high impact publications to warrant increased funding. The proposed budget is rather high relative to track record, and the PI is reliant upon the Chembridge libraries to be successful. The target identification section is very weak. To raise the level of enthusiasm, the PI should limit the proposed studies to the first 2 Aims, and develop these in a much more comprehensive and detailed manner. DISCUSSION: The applicant proposes a chemical genomics screens for pluripotency. Non-NIH lines will be used since they are more readily manipulated for single cell analyses than are approved lines. This approach attempts to address the need to find maintenance factors for hESCs, and these studies would be expected to yield important information. A main concern of reviewers is the lack of productivity of this investigator, who has no publications in high impact journals. The proposal was considered an overambitious "fishing expedition." One reviewer noted that although the degree of innovation in this proposal is not great, the unique environment in San Diego leads the reviewer to believe that these studies will be successful. Another reviewer found that the studies proposed were simply too ambitious based on the record of productivity of the PI. This reviewer felt that the investigative team would be unlikely to complete the proposed work. The reviewer also had doubts about the ChemBridge libraries, believing that they have a number of flaws and stated that other sources should have been mentioned as alternatives.

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