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RS1-00188-1: DETERMINING THE ROLE OF MITOCHONDRIA IN THE REGULATION OF HUMAN EMBRYONIC STEM CELLS

Recommendation: Not recommended for funding

Public Abstract (provided by applicant)

The expectations for stem cell transplantation to treat diseases have been high. However, accumulating evidence indicates that stem cell homing is a very tough issue, and stem cell transplantation seems to result in a majority of “homeless stem cells”. Stem cells’ innate ability to travel to the right place in the body is very limited. Only a very small fraction of cell grafts will dock properly. The rest will be eliminated by apoptosis or become problematic. On the other hand, human embryonic stem cells (hESCs) are notoriously difficult to maintain due to spontaneous differentiation and apoptosis within culture. Understanding mechanisms that regulate hESC self-renewal, differentiation, and apoptosis will enhance our ability to manipulate hESCs and to realize their potential for regenerative medicine. Previous studies on the regulation of stem cell behavior have focused largely, if not exclusively, on the role of nuclear genes. The present proposal seeks to expand this existing paradigm. We hypothesize that mitochondria play a critical role in determining stem cell fate. We propose to determine the role of mitochondria in the regulation of hESC behavior with a combination of cellular and molecular techniques to provide overlapping approaches to unravel novel mechanisms controlling hECs survival, self-renewal and differentiation. We will test whether the mitochondrial fusion and fission machinery regulates mitochondrial morphology and redox state, and thus play important roles in hESc fate choices. This proposal will reveal important mitochondrial signaling pathways in regulating hESCs. The information will expand our ability to manipulate hESCs, with the ultimate goal of designing efficient hESC-based therapies.

Statement of Benefit to California (provided by applicant)

The state of California is a pioneer in supporting critically important stem cell research. Our research will enhance our ability to propagate and culture these cells and advance our understanding of the mechanisms that mediate stem cell development and differentiation. This work will provide new insights into mitochondrial mechanisms that regulate hESCs and potential strategies for treating mitochondrial diseases that may directly affect Californians suffering from such diseases, for which there currently is no cure.

Review

SYNOPSIS OF PROPOSAL: This proposal is will test the hypothesis that mitochondria play a critical role in determining stem cell fate. The proposal contains three specific aims. First, the investigator will attempt to determine whether the mitochondrial activities regulate hESC pluripotency. The second specific aim is to determine the role of mitochondrial activities in other hESCs processes. The third specific aim tests where mitochondrial machinery regulates the hESC activities. Despite what is stated by the author, this work is fundable by federal and other sources of funding.

INNOVATION AND SIGNFICANCE: As the role of mitochondria in any system or cell type is under-explored, any additional knowledge gained, including that proposed by this application, will inevitably have an impact. While the approach is not particularly hypothesis-driven, it may be significant in that it seeks to uncover the role of mitochondrial dynamics in hESC behavior.

STRENGTHS OF THE PROPOSAL: In all aims the experiments are nicely designed and well controlled, and the diagnostic of outcome is based on the expression of markers.

The Principal Investigator (PI) shows some Preliminary Data from pilot studies demonstrating that there is a good chance the proposed experiments can be carried out successfully. The Specific Aims and Experimental Approach in the application are logical, sequential, and relatively well thought through. However, details of the proposed analysis are inadequate. The PI has a strong background and successful track record in neither hESC biology nor mitochondrial biology. He/she relies on team members and collaborators for those expertise. This is a potential problem.

WEAKNESSES OF THE PROPOSAL: While the role of mitochondrial actvities can be studied in any cell type, it’s not very clear why this would represent a priority for hESC work. In addition, all the experiments are to be performed on one line, which is on the NIH registry. Enthusiasm was therefore lowered on the grounds that it was difficult to appreciate why this was important to undertake at this time on this particular cell line using CIRM funds.

The PI and team assembled for this project seem to lack the necessary expertise. Additionally, another weakness of this proposal is its lack of an overall hypothesis. These genes proposed for study have been implicated in the control of mitochondrial activities. The proposed work is eligible for NIH-funding. The PI proposes to use an NIH-eligible hESC line.

The PI only designates 5% of his time to this project. In addition to the PI, there is a Postdoctoral fellow as Co-PI committed at 20% and another Postdoctoral fellow committed at 6%. It is very difficult to imagine how the work proposed in this application will be completed by less than 1/3 of an FTE.

A few other problems were found with this proposal. In the Oversight section – this proposal does NOT use human oocytes, human in-vitro embryos, or derive a covered stem cell line. This project does NOT require IRB approval, nor does it involve the use of human subjects. This project does NOT require the use of vertebrate animals and does NOT require IACUC approval. However, the project does use hESC lines and these cells should be considered biohazardous materials as they are human tissue.

There were also many issues with the Budget as submitted, including the amounts budgeted for supplies and travel and their justification. Any experienced hESC researcher performing similar experiments would understand that each supply category has been severely over-budgeted.

DISCUSSION: There is no strong hypothesis here and the analyses are not well-described. Only one line will be studied (which is NIH-fundable), and the team lacks expertise. Since mitochondria can be studied in any cell type, this work is not a priority for CIRM funding. A reviewer also commented that the time allocation under “percent effort” listed in the proposal adds up to less than 1 FTE.

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