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RC1-00100-1: Functional Genomic Analysis of Chemically Defined Human Embryonic Stem Cells

Recommendation: Recommended for funding
Scientific Score: 81

First Year Funds Requested: $642,500.00
Total Funds Requested: $2,570,000.00

Public Abstract (provided by applicant)

Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. Because DNA damage occurs during normal cellular proliferation and can cause DNA mutations leading to genetic instability, it is critical to elucidate the mechanisms that maintain genetic stability during self-renewal. This is the overall goal of this proposal. Based on our recent findings, I propose to investigate two major mechanisms that might be important to maintain genetic stability in hESCs. First, I propose to elucidate pathways that promote efficient DNA repair in hESCs. Second, based on our recent findings, I hypothesize that another primary mechanism to maintain genetic stability in self-renewing hESCs is to eliminate DNA-damaged hESCs by inducing their differentiation. Therefore, I propose to identify the pathways that regulate the self-renewing capability of hESCs in the presence and absence of DNA damage. In summary, the proposed research will contribute significantly to our understanding of the pathways important to maintain self-renewal and genetic stability in hESCs. This information will provide the foundation to improve the culturing condition of hESCs to promote efficient self-renewal with minimum genetic instability, a prerequisite for the development of hESCs into human therapeutics.

One major objective of the proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. While mouse models are valuable tools to study the mechanisms of the pathogenesis in human diseases, many differences between mouse and human cells can lead to distinct phenotypes as well as the common phenomenon that certain therapeutic interventions work well in mouse models but poorly in humans. Therefore, it is of high priority to create disease-specific hESCs as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to employ the transgenic and gene targeting techniques to create disease-specific hESCs. Therefore, the proposed research will significantly improve our capability to generate disease-specific hESCs. After experimenting with various existing hESC lines, we found that only the non-federally-approved hESC lines developed recently at Harvard University is most suitable for genetic manipulation technologies. Since the research involving the HUES lines can not be supported by federal government, CIRM is in a unique position to support this proposed research.

Statement of Benefit to California (provided by applicant)

Human embryonic stem cells (hESCs) are capable of unlimited self-renewal, a process to reproduce self, and retain the ability to differentiate into all cell types in the body. Therefore, hESCs hold great promise for human cell and tissue replacement therapy. The major goal of the human stem cell research supported by proposition 71 is to improve and even realize the therapeutic potential of hESCs. DNA damage occurs during normal cellular proliferation of hESCs and can cause genetic mutations that will be passaged to derivatives. Any cells with genetic mutations are not suitable for therapeutic purpose since they can cause cancers in the recipient. Therefore, to achieve the therapeutic potential of hESCs, it is critical to elucidate the mechanisms that prevent genetic mutations during the self-renewal of hESCs. This is the overall goal of this proposal. Successful completion of the proposed research will help to optimize the culturing conditions that promotes efficient self-renewal with minimum genetic instability.

One high-priority area of hESC research is to create disease-specific hESCs, which can be used as powerful genetic tools to study the mechanism of the pathogenesis in human diseases. In addition, the unlimited supply of primary cells derived from the disease-specific hESCs will become valuable reagents for drug discovery. There are two ways to generate the disease-specific hESCs. One approach is through nuclear transfer that has been proven extremely difficult in human context and so far unsuccessful. The other is to develop the transgenic and gene targeting techniques to create disease-specific hESCs. One major objective of my proposed research is to improve the genetic manipulation technologies in hESCs, including transgenic and gene targeting technologies. The successful completion of the proposed research will significantly improve our capability to generate disease-specific hESCs. In addition, the disease-specific hESCs (ATM-/- and p53-/- hESCs) generated in the course of the proposed studies are valuable tools to study the basis of neuronal degeneration in Ataxia-telangiectsia and development of human epithelial tumors as a result of p53-deficiency. Both of these phenotypes are not observed in mouse models.

In summary, the proposed research will benefit California citizens by contributing to the eventual realization of the therapeutic potential of hESCs.

Review

SYNOPSIS: DNA damage such as double-stranded breaks (DSBs) occurs during normal cellular proliferation. The main goal of this proposal is to elucidate mechanisms by which human Embryonic Stem Cells (hESC) maintain self-renewal and genetic stability. There is recent evidence from this group that DNA damage induces differentiation of murine Embryonic Stem cells (ESC) by suppressing Nanog expression. The Principal Investigator (PI) hypothesizes that one mechanism to maintain genetic stability of self-renewing ESCs is to induce the differentiation of DNA-damaged ESCs. The proposal includes relevant preliminary data.

IMPACT & SIGNIFICANCE: This proposal’s importance rests on its focus on how human ES cells (hESCs) are maintained in a pluripotent stem cell state. It addresses the potential role of DNA damage responses in promoting differentiation and hence elimination of cells from the stem cell pool. One key hypothesis in stem cell biology is that stem cells have evolved multiple mechanisms to maintain the integrity of the stem cell pool, from export of potentially toxic molecules via strong pumps (MDR, etc.) to extremely active DNA repair mechanisms. This proposal seeks to understand the hESC response to DNA damage, and proposes to explore the finding that DNA damage triggers departure from the hESC pool by down-regulation of Nanog to uncover the molecular players in the DNA damage response.

The proposed research has potential to impact cancer biology and stem cell biology by revealing how transcription factors affect responses to DNA damage in hESC. Generation of knockout hESC lines also provides reagents for future examination of transcription factor function in specific human cell lineages.

QUALITY OF THE RESEARCH PLAN: Reviewers all agree that the overall quality of the research plan is quite logical, well laid out, and generally very strong. The project will yield important information, both for the hESC field as well as cancer biology in general. The PI and his/her collaborators are experts in this sort of research. The research team includes includes an expert collaborator for mass spectrometry, another who is a systems biologist, another who discovered the transcription factor link and is first author of an important paper in this research area that was published in a high profile journal, and another who studied post-translational modifications of one of the relevant transcription factors as a doctoral student. The timetable for achieving their goals is reasonable. Most of the aims are backed by solid preliminary data supporting both the feasibility and rationale of the approaches. The PI has established methods in the laboratory for transfecting non-NIH approved hESCs (NIH-approved lines grow as clumps). Importantly, the development of homologous recombination-based modification of hESC genes allows for simple and powerful experimental approaches.

The experimental plan consists of 3 specific aims. The first aim is to determine the roles of a key transcription factor in DNA damage responses in hESCs. The investigators will use Chip to see if this factor binds nanog promoter; and propose to make knockout hESCs by sequential targeting (although a reviewer questions the practicality of sequential targeting in hESCs). The PI will test nanog RNA levels before and after DNA damage then study checkpoints. hESCs have robust S and G2/M checkpoints after DNA DSB damage but undergo apoptosis after UV exposure. A key trasncription factor is required for the G2/M checkpoint in human but not mouse cells. The approaches proposed to address the role of the factor in maintaining genetic stability of hESC follow previously successful approaches performed with murine ESC. Creation of the knockout hESC line will provide a valuable reagent for assessing the role of the transcription factor in hESC. These approaches are supported by preliminary data showing Nanog expression is reduced in hESCs following DNA damage and the ability of forced expression of Nanog to rescue mESC self-renewal following exposure to ionizing radiation.

The second aim is to define the role of nanog in self-renewal in the presence and absence of DNA damage. The investigators will look for associated proteins in the presence and absence of DNA damage; they will tag nanog and use immunoprecipitation and sequencing to identify associated proteins. They will study candidates functionally by blocking, and define interactions with nanog. No clear rationale is given for examining differences in Nanog-interacting proteins before vs. after DNA damage. The reason this is problematic is based on the applicants preliminary data. Therefore, while going after Nanog protein interactions is a fine goal in itself, going after Nanog protein interactions specifically in response to damage appears to provide a less worthwhile activity.

The third aim is to study DNA damage response pathways important for genetic stability of hESCs; another transcription factor is activated in hESCs after DNA damage. The investigators will generate knockout cells and knockin cells. The PI claims to have one potential positive for the knockin among 200 colonies screened. This aspect of the proposal entails sequential gene targeting, again, with questions as to feasibility. The PI will study pathways to activation and proteins involved in sensing DNA DSB in hESCs, and also will use a DS-DNA pulldown assay to identify a protein complex that is specifically associated with DNA DSBs in hESCs and fibroblasts. Candidates will be studied as needed. The generation of knockout and knockin hESC lines offers some very interesting possibilities. Simple, logical and meaningful experiments are proposed after knockout clones are obtained.

STRENGTHS: The PI proposes interesting and clear hypotheses that might be relevant to maintaining self-renewal and optimizing hESC cultures. S/he provides strong preliminary data in support of the proposal, first in murine ESC and now in hESC. S/he apparently has found conditions for the genetic manipulation of hESCs, including possibly for gene targeting (at least one example).

The research team assembled is strong, and includes a protein sequencer and systems biologist.

This is considered a well organized proposal, with a clear and logical plan of attack. The requisite reagents and skills are in hand and preliminary data support the feasibility of most approaches.

The results generated should be important, and could be useful in many scientific venues. Knockout cell lines likely will be valuable reagents for experiments beyond the scope of this proposal.

WEAKNESSES: One weakness is the questionable feasibility of making double-targeted knockout hESCs. Much of the work described depends on the successful derivation of these lines. Proposing experiments with reagents such as knockout cell lines that have not yet been generated is considered somewhat risky.

A clear rationale for Aim 2 is absent. An additional weakness noted is that the functional testing of candidates is rather sketchy in the application.

One reviewer states that in some sense, this sort of research does not necessarily need to be done in hESCs right now; murine ESC results should be just as interesting, and perhaps more tractable since the assays for self-renewal and function have worked out better.

A reviewer recommends that the PI generate plans on how to study candidates that emerge from the proteomic experiments in Aim 2.

DISCUSSION: Reviewers agree that this is an interesting proposal, described by one reviewer as ‘elegant’, and the PI has assembled an excellent team. Of particular note is that the PI has worked with and established transfection methods using a non-presidential hESC line. One reviewers enthusiasm was dampened by the view that, strictly speaking, the proposed questions could easily have been addressed in the murine ESC system. Another reviewer would have liked to see more rationale, (e.g. why look at nanog before and after DNA damage), and concerns were expressed regarding the feasibility of the double targeting strategy.

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:

• Lansing, Sherry