Funding opportunities

Mitochondrial Dysfunction in Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00432
Principle Investigator: 
Funds requested: 
$632 500
Funding Recommendations: 
Recommended
Grant approved: 
Yes
Public Abstract: 
Mitochondrial Dysfunction in Embryonic Stem Cells {REDACTED} A major concern for the utilization of human Embryonic Stem Cells (hESCs) for cell replacement therapy is that with prolonged culture, the capacity of the cells to generate the desired cell types for therapy declines. While the reason for this is currently unknown, our research suggests that an important factor is damage to the genetic blueprints that are necessary to sustain the cellular power plants of the cell, the mitochondria. The human cell is the product of a symbiotic merger that occurred two billion years ago of two different cell types: one generating the host cell and the other generating an intra-cellular colony of bacteria, the mitochondria. In the modern human cell, the host cell constitutes the nucleus and the cytosol and the genetic information (DNA) for this nucleus-cytosol organism resides in the nucleus and is responsible to building and maintaining the structural elements of the cell: analogous to the carpenters blueprints for building a house. The mitochondria have their own DNA blueprints, the mitochondrial DNA (mtDNA), and this describes the circuit diagram for the energy production system of the mitochondria: analogous to the electrician’s wiring diagram for the house. Mutations which damage the mtDNA circuit diagram result in the mitochondria’s inability to repair damage to the mitochondrial energy production system. As the efficiency of the mitochondrial power plants declines, they make less energy and more smoke, the smoke generated being oxygen radicals. As oxygen radical production increases, it causes increased damage to the mitochondria and mtDNAs, ultimately resulting in the mitochondrial power plants go off-line and the death of the cell. As mitochondrial oxygen radical production increases, it stimulates the cell to divide in an effort to dilute out the smoke generating mitochondria. However, the problem is that the damaged mtDNA blueprints replicate along with the cell. Hence, the toxicity continues to increase. We and others have documented this type of phenomenon occurs in a variety of cultured cell types. Therefore, it is likely that it also occurs in hESCs. If so, as damage to the mtDNA accumulates, hESC energy production declines and oxygen radical production increases until the hESC is no longer capable of building the more complex structures necessary to create tissue replacement cells. If we can prove that this scenario does occur in hESCs, then we can develop drugs that will limit mitochondrial oxygen radical production and protect the mitochondria and mtDNAs from oxygen radical damage. Furthermore, we have developed a method that permits us to replace damaged mtDNAs in cells with new one. Hence we could repair the mtDNA damage of aging hESCs and regenerate their capacity to make high quality differentiated cells for use in tissue replacement therapy.
Statement of Benefit to California: 
Prolonged cell culture of human Embryonic Stem Cells (hESCs) frequently results in the loss of the cell’s capacity to differentiate on command into well differentiated cells. This eliminates their utility for generating replacement cells for use in cell replacement therapy to repair damaged tissues and organs within the body. The reason for this loss of developmental capacity by the hESCs is currently unclear, but we believe that a major factor contributing to the decline in the therapeutic value of hESCs is the accumulation of deleterious mutations in the mitochondrial DNA (mtDNA) of the cultured hESCs. The mtDNAs are located in the mitochondria which are organelles in the cytoplasm of the human cell. The mitochondria are responsible for generating most of the energy used by the cell and as a toxic by-product, the mitochondrial generate most of the endogenous reactive oxygen species (ROS). The mtDNA encodes key elements of the mitochondrial energy generating apparatus, and since ROS is a mutagen, the mtDNA is highly prone to acquiring mutations in these energy genes. These mutations then inhibit mitochondrial energy production which also results in increased ROS production. Increased mitochondrial ROS production stimulates the cell growth, so the cells with the mutant mtDNAs out grow the normal cells. However, the more rapidly growing cells with the mutant mtDNAs also have reduced mitochondrial energy production, which together with the increased ROS production, inhibits the developmental capacity of the hESC.In this research, we propose to establish that deleterious mtDNA mutations do in fact accumulate in hESCs over time and that they play an important role in the loss of the developmental potential of hESC cells. If we can confirm that this is a fact, then we should be able to greatly increase the therapeutic potential of hESCs by developing procedures for protecting the mtDNA of the hESCs from oxidative damage. This might be accomplished by growth of hESCs in the presence of mitochondrially target antioxidants. Furthermore, cells that had lost their developmental potential might be revitalized by simply replacing the damaged mtDNAs with good mtDNAs using our trans-mitochondrial cybrid technique.Thus, the proposed research has the potential of greatly increasing the therapeutic potential of all hESCs that will be developed in the State of California.
Review Summary: 
SYNOPSIS: The proposal focuses on a much-neglected area of hES cell biology, the mitochondria. The aims are to (1) create hESC that contain mtDNA mutations that cause human disease and examine their growth and differentiation into neurons and (2) analyze the mtDNA mutations in various hESC lines and the effect of these mutations on growth and differentiation. SIGNIFICANCE AND INNOVATION: The innovation is high in this proposal, bringing experts in the mitochondrial medicine field into hES cell biology. The investigators choose to look at the mitochondrial genome in hES cells and assess its importance in pluripotency and differentiation, a subject that almost no one has had the foresight to examine. This will be an exhaustive look at mitochondrial function in hES cells, and hopefully this information will be used down the road to help increase the efficiency of cell cultivation techniques and the efficiency of some differentiation pathways. Unfortunately, as there is no strong evidence that mitochondrial mutations effect hES pluripotency the significance of the proposal is unclear, but potentially quite high. STRENGTHS: The strength of the proposal is the enormous expertise in mitochondrial medicine and biology brought to the table. The investigators and their collaborators are clearly among the world’s leaders in understanding mitochondrial function and its impact on cell biology. One reviewer stated this is hands down the most well written, well planned and well substantiated proposal they have the opportunity to evaluate - one word, excellent. WEAKNESSES: One weakness of the proposal is that there is no evidence that mutations in the mitochondrial genome exist in ES cells or that they will effect pluripotency, but that is all the more reason to do the experiments. Another weakness is that the description of neuronal differentiation, and particularly the analysis of neuronal differentiation, is left to the imagination. Presumably non-neuronal differentiation will be examined as well as looking for the efficiency of neuronal differentiation. In this regard, ‘differentiation’ is treated as a single entity in the grant, and it is likely based on the literature, that differentiation will be skewed (not just halted) by changes in mitochondrial function. Reviewers had three suggestions for the PI. First, the aims should be reversed in time. It would be good to know the mitochondrial history of the cells before they are used for the heteroplasmic ES cell substrates. Second, it is clear that hES cells generated by SCNT would display considerable mitochondrial heteroplasmy, which might effect their growth, viability, pluripotency, and utility for cell transplantation (i.e., rejection due to donor oocyte mitochondria). The investigators might mention this as a nice benefit of doing these types of experiments in their proposal. Third, the investigators might create a seemingly normal mitochodrial heteroplasmic hES cell and assess the consequences. DISCUSSION: This proposal was one of the best that was reviewed by the panel. The work may not be terribly sexy, but looking at the effects of mitochondrial mutations on growth and differentiation is incredibly innovative. The science is beautifully presented, and SCNT approach is "incredibly innovative", especially in studies with mitochondria and pluripotency. To make this totally great, the PI would only have to reverse the order of the Aims in time.
Conflicts: 

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