Funding opportunities

Ubiquitin-dependent control of hESC self-renewal and expansion

Funding Type: 
Basic Biology III
Grant Number: 
RB3-02222
Principle Investigator: 
Funds requested: 
$1 364 091
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) provide an invaluable resource for regenerative medicine and disease modeling. To be able to use these cells in the clinic, hESCs and iPSCs need to be expanded without introducing genetic instability. However, current protocols of hESC and iPSC propagation frequently result in aneuploidy, a potentially tumorigenic cell state. Because of their tumorigenic potential, undifferentiated hESCs have to be removed from cell populations prior to transplantation, yet efficient ways to securely achieve this have not been developed. Together, these limitations greatly limit the use of hESCs or iPSCs in regenerative medicine. Here, we propose to dissect and manipulate mechanisms of hESC division and survival. Based on our preliminary data and previous observations in embryonic cells, we will initially dissect the role and regulation of the anaphase-promoting complex (APC/C), an essential component of the core cell cycle machinery, and Cul3, an enzyme required for the integration of extracellular signaling into the hESC division program. These experiments will make use of our experience in developing biochemical systems to dissect complex pathways in vitro, combined with an in-depth analysis of cell cycle control in hESCs in vivo. Understanding hESC division control by APC/C and Cul3 will identify the mechanisms generating aneuploidy during hESC culture. Subsequently, we will isolate novel hESC-specific ubiquitination enzymes required for division and survival by using siRNA screens in hESCs. We will identify the substrates of critical enzymes to determine their role in division and survival control. Manipulating the activity of hESC-specific enzymes or substrates will allow the removal of undifferentiated cells from cell populations, an essential step prior to transplantation. The results from these studies will provide critical insight into the mechanisms controlling hESC division and survival. Based on findings on division control, we will be able to develop protocols for faithful hESC or iPSC expansion in culture, while understanding the mechanisms of hESC survival will point to strategies for the selective elimination of undifferentiated cells from cell populations. Both outcomes of this study will greatly expand the use of stem cells for regenerative medicine.
Statement of Benefit to California: 
Human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) provide an invaluable resource for regenerative medicine and disease modeling. To be able to use these cells in the clinic, hESCs and iPSCs need to be expanded without introducing genetic instability. However, current protocols of hESC and iPSC propagation frequently result in aneuploidy, a potentially tumorigenic cell state. Because of their tumorigenic potential, undifferentiated hESCs have to be removed from cell populations prior to transplantation, yet efficient ways to securely achieve this have not been developed. Together, these limitations greatly limit the use of hESCs or iPSCs in regenerative medicine. Here, we propose to dissect and manipulate mechanisms of hESC division and survival. We will identify and dissect the mechanisms that control the core hESC division machinery and those that achieve the integration of extracellular signals into the hESC division program. Findings from these studies will allow us to develop protocols for the faithful expansion of hESCs or iPSCs in culture, an essential step for using these cell types for differentiation or as disease models. In the last part of this work, we will use siRNA screens to isolate novel ubiquitination enzymes and their substrates that are required for hESC division and survival. Manipulating the activity of these proteins will provide strategies for eliminating undifferentiated cells from cell populations. As current differentiation protocols are inefficient, the selective removal of undifferentiated cells is required before transplantation of differentiated cells can occur. The results from this part of our study can also be implemented to selectively kill de-differentiated tumor cells during chemotherapy. Together, our study will provide critical insight into the mechanisms controlling hESC division and survival to develop protocols for faithful hESC or iPSC expansion in culture and for the selective elimination of undifferentiated cells from cell populations. Both outcomes of this study can be directly translated for applications in the clinic, greatly expanding the use of stem cells for regenerative medicine.
Review Summary: 
Project Synopsis: This proposal aims at determining the role of ubiquitination in controlling cell division and survival of human embryonic stem cells (hESCs). It is based on the rationale that misregulation of the anaphase-promoting complex (APC/C), a ubiquitination enzyme involved in proper chromosome segregation, leads to aneuploidy, a common phenomenon occurring in hESCs. The goal of the first aim is to dissect the mechanisms by which APC/C controls cell division in hESCs. In the second aim, the applicant proposes to analyze how another ubiquitination enzyme, Cul3, integrates extracellular signaling into the hESC cell division program. Finally, Aim 3 consists of a screen for novel ubiquitination enzymes required for hESC proliferation and survival. Significance and Innovation: - This proposal addresses a major unsolved problem in stem cell biology, a lack of high fidelity methods for expansion of genetically stable human pluripotent stem cells (hPSC). As both aneuploidy and unchecked self-renewal increase the risk of tumorigeneis, this project, if successful, could have a major impact on the potential application of stem cells for regenerative medicine. - While some reviewers did not feel that further delineation of cell cycle control in hESCs represents an urgent priority, others considered this proposal highly significant for its focus on two understudied aspects of pluripotent stem cell biology: 1) the unique cell cycle of hPSC compared to differentiated cells; and 2) the role of ubiquitination in controlling cell division and survival. - It remains unclear if the long-term outcomes anticipated by the applicant (i.e. the development of improved hESC culture conditions leading to less aneuploidy and strategies for selectively eliminating undifferentiated cells from differentiated populations) will be achieved. However, this is a sound project with a rigorous approach toward a mechanistic understanding of hESC cycle control that should lead to generation of new data. - While some aspects of the work are not particularly innovative, Aim 3 is both creative and daring. Feasibility and Experimental Design: - The aims of the proposal are logically presented in a detailed manner. - Previous work by the investigator impressively demonstrates that the proposed approaches have been useful in dissecting the role of the ubiquitination enzymes and substrates in somatic cell types and in mouse (m)ESCs. However, there is a lack of preliminary data using hESCs. Given the significant differences in the ease with which mESCs and hESCs are handled experimentally, this type of preliminary data would have alleviated any concerns about the ability of the investigators to conduct the proposed studies. - In light of major known differences between mESCs and hESCs signaling pathways mediating pluripotency, extrapolating from preliminary data collected using mESCs might be misleading. - Aim 2 does not consider that the signals under study act as morphogens, eliciting different outcomes at different thresholds or duration of signaling, rather than acting as binary switches. - A minor flaw is the limited discussion of potential pitfalls and alternative approaches that might address them. Principal Investigator (PI) and Research Team: - The applicant is an assistant professor with a strong track record of success and a number of outstanding publications in top tier journals. - The research team has much of the required expertise to conduct the proposed research. The one missing component is any documented evidence that they can maintain or manipulate hESCs. - The resources for the proposed studies are appropriate. - The budget is appropriate. Responsiveness to the RFA: - This proposal is within the scope of the RFA as it is entirely focused on elucidating basic molecular and cellular mechanisms of hESC proliferation and survival.
Conflicts: 

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