Basic Biology II
The main objective of the CIRM Basic Biology Awards II is to provide funding for cutting-edge stem cell research and to tackle significant unresolved issues pertinent to understanding the biology of human embryonic stem cells and the control of stem cell fate. Our laboratory recently discovered that we can modulate the expression of genes that regulates cell fate by activating a key factor (Akt) in the power plant (mitochondria) of human embryonic stem cells. This is a novel mechanism that has not been described in the human embryonic stem cells. Potential ability to adjust cell fate through modulation of energy production will add a novel method to interrogate and manipulate the regulatory network that defines the identity of human embryonic stem cells. The proposed studies will investigate how Akt interacts with other molecules in the power plant in human embryonic stem cells, how this new paradigm regulates energy production, and how such mechanism regulates stem cell fate and survival. Future therapeutic application of stem cells will rely heavily on delicate control of cell fate. We will test our hypothesis that human embryonic stem cell gene expression and cell fate can be altered by adjusting the power plant of stem cells. The results of this project will provide opportunities to identify new targets that can be used to manipulate embryonic stem cell fate in the future, and will advance our understanding of the relationship between regulation of energy production and stem cell fate.
Statement of Benefit to California:
A primary goal of CIRM is to translate basic stem cell research to clinical applications. The disability and loss of earning power and personal freedom resulting from a disease or disorder are devastating and create a financial burden for California in addition to the suffering caused to patients and their families. Therapies using human embryonic stem cells (hES cells) have the potential to change millions of lives. For the potential of hES cells to be realized, we have to decode basic mechanisms that direct stem cell development into specialized cells. Future application of stem cells will rely heavily on delicate control of cell fate. Potential ability to adjust cell fate through modulation of cellular signaling will add new method to manipulate the regulatory network that defines the identity of human embryonic stem cells. Potential benefits of this project to the Citizens of California include: 1. Development of new methods based on modulation of mitochondria function to direct the stem cell fate and stem cell viability, which may eventually be used to develop new strategies to design cell replacement therapies for human diseases. 2. Systemically screening and identifying new protein targets that may be used to develop new drugs and agents to promote stem cell function, thereby developing new treatment methods. 3. Transfer of new technologies and intellectual property to the public realm with resulting IP revenues coming into the state. 4. Creation of new biotechnology spin-off companies based on generated intellectual property. 5. Creating interdisciplinary research teams that will have a competitive edge for obtaining funding from out of state. 6. Creation of new jobs in the biotechnology sector.
EXECUTIVE SUMMARY This proposal is concerned with the regulation of human embryonic stem cell (hESC) function by mitochondria, the cellular organelles of energy generation. The study will focus on the role of mitochondrial translocation of Akt1, a protein kinase and key signaling component, and investigate how this translocation affects respiration, cell fate, and survival of hESCs. The first specific aim is to characterize Akt1 translocation to mitochondria in hESCs and identify mitochondrial targets of Akt1 signaling. The second specific aim will be to analyze effects of Akt1 translocation on oxidative phosphorylation and respiration. The third aim is to determine the effects of Akt1 activation on stem cell gene expression, proliferation, apoptosis, and differentiation. The project explores an important aspect of stem cell biology, signaling pathways that regulate cell fate decisions. However, reviewers were unconvinced about the specific significance of mitochondrial association of Akt1 and the proposed project overall. In particular, compelling preliminary data indicating that mitochondrial translocation of Akt1 actually occurs in hESC and that this event has important consequences for stem cell fate are lacking. Additionally, conflicts between supporting data published by the applicant and those reported by another group concerning various forms of Akt1 created further doubts about the project’s significance and potential impact. Reviewers expressed serious concerns about the project’s feasibility. Although they recognized the novelty of the applicant’s hypothesis and noted that many experiments were based on established methodologies, they found significant flaws in the experimental approach. In particular, much of the proposed work is base on over-expression of a targeted, constitutively active form of Akt1, and there is little evidence that behavior of this exogenous protein will mirror the endogenous activity. Many experimental details and the rationale for specific approaches raised further concern. For example, it was unclear why serum-deprivation would be used to monitor Akt1 translocation or why an extended time-course with continuous activation of Akt1 is proposed for transcriptional profiling experiments. Reviewers found the preliminary data to be unconvincing. They further viewed Aim 3 as unfocused and unlikely to provide critical information about the regulation of cell fate. The PI has considerable expertise in metabolism and cardiology but lacks substantial experience in stem cell biology. Some concern was expressed about the applicant’s modest publication record and lack of other, significant research support. Named collaborators and the research environment were judged to be excellent. In summary, the proposed research will study consequences for stem cell fate of mitochondrial association of the signaling component Akt1. Strengths of the proposal include its novel hypothesis and the importance of its broad goal in elucidating regulatory mechanisms in hESC. Weaknesses include lack of compelling preliminary data and significant flaws in the experimental design.