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:
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.
SYNOPSIS: This proposal is based on testing the hypothesis that mitochondria play a critical role in determining stem cell fate. Specifically, the author proposes to test how mitochondrial fusion and fission regulate mitochondrial morphology and redox state and how it may be involved the in the choice between self renwall differenat and apoptosis in hESCs. Despite what is stated by the author, this work is fundable by federal and other sources of funding. The proposal contains three specific aims. First, the investigator will attempt to determine whether the mitochondrial fusion and fission regulate hESC pluripotency. The second specific aim is to determine the role of mitochondrial fusion and fission in apoptosis in hESCs. The third specific aim tests the hypothesis that mitochondrial fusion and fission machinery regulates the hESC fate choice by controlling mitochondrial morphology and redox state.
SIGNIFICANCE AND INNOVATION: 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 a strong impact. There is therefore a general positive significance to this type of approach.
The PI proposes to study the role of mitochondrial fission and fusion on pluripotency, apoptosis, and fate choice in one hESC line. While the approach is not particularly hypothesis driven, it is innovative. The proposal may be significant in that it seeks to uncover the role of mitochondrial dynamics in hESC behavior.
STRENGTHS: In all aims the experiments are nicely designed and well controlled, and the diagnostic of outcome is based on the expression of markers.
The PI show 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, the proposed analysis is inadequate. For instance, the PI proposes to analyze the hESCs based on marker expression but does not discuss the number of cells, number of passages, and other parameters affecting the analysis. The PI has a strong background and successful track record in neither hESC biology nor michondrial biology. He relies on team members and collaborators for those expertise. This is a potential problem.
WEAKNESSES: While there is nothing scientifically wrong with this proposal, it is difficult to get excited about this grant because of the lack of significance relevant specifically to hESCs. While the role of mitochondrial fusion and fission can be studied in any cell type, including progenitor cells, 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 (H1 line). 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. The PI generally proposes to investigate the role of mitochondrial fusion and fission in hESC behavior through the action of two genes – Mfn2 and Drp1. These genes have been implicated in the control of mitochondrial fusion and fission. The proposed work is eligible for NIH-funding. The PI will use the NIH-eligible hESC line H1.
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 a few issues with the Budget: the amount budgeted for travel seems high. Using the travel budget for the Neuroscience and other meetings does not seem justified. There is no mention of travel to the annual CIRM meeting. The PI requested $140,000 for supplies. This was broken down to cell culture, immunocytology, biochemistry, and other categories. This amount of money for an annual supplies budget is extraordinary and unjustified. 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 work), 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.