Proteomics of the Oxidative Stress Response in Embryonic Stem Cells

Funding Type: 
SEED Grant
Grant Number: 
RS1-00434
ICOC Funds Committed: 
$0
Stem Cell Use: 
Embryonic Stem Cell
Public Abstract: 
The oxygen we breathe from the air can be toxic if cells are exposed to high levels. One-fifth (20%) of the air we breathe is oxygen, but as it filters through our blood to our body’s tissues the level drops to as low as 2% and most cells in the human body including the stem cells are exposed to this level of oxygen. If cells are exposed to high levels of oxygen it creates something called “oxidative stress” that leads to rapid cellular aging and the related loss of physiological function. This is thought to result from the progressive accumulation of oxidative damage to proteins and the DNA. Evidence is emerging that embryonic stems cells are exquisitely sensitive to oxidative stress. Routine isolation and maintenence of human embryonic stem cell lines is typically performed in humidified air (20% oxygen) in incubators kept at body temperature. Maintenance of human embryonic stem cells in 2% as opposed to 20% oxygen increases their proliferative lifespan and alters the profiles of genes that influence the conversion of human embryonic stem cells into useful blood forming cells. If human embryonic stem cells are to be useful therapeutically we will need to grow them effectively in culture and optimize the conditions for their expansion. In the present study we will grow human embryonic stem cells in 2% versus 20% oxygen and study their protein content or profile using a new technology called “proteomics”. This allows one to look at many proteins at once and quickly identify differences between two differently cultured sets of cells. We will also study the response of human embryonic stem cells to chemically-induced oxidative stress. These studies will characterize for the first time the oxidative stress response of embryonic stem cells and their progeny at the protein level. This information could be critical to the successful culture and expansion of human embryonic stem cells for therapeutic purposes. Further, understanding how embryonic stem cells respond to oxidative insults has implications not only for how we isolate and identify therapeutically useful stem cell lines, but also directly adds to our understanding of how environmental chemical exposures can impact our health. Leukemias are a disease of the blood forming stem cells that can be induced by pro-oxidant chemical exposure. Defining at the protein level how stem cells respond to oxidative stress may identify early markers of disease or exposure.
Statement of Benefit to California: 
Understanding how human embryonic stem cells (hESCs) respond to toxic insults has implications not only for how we isolate and identify therapeutically useful stem cell lines, but also directly adds to our understanding of how environmental exposures to chemical can impact our health. The potential of hESCs to both self renew and differentiate is seen as a boon for tissue engineering and cell therapy, particularly in regard to the hematopoietic or blood forming compartment. California’s Biotechnology industry is already at the forefront in developing therapeutics based on cell therapy and is poised to further benefit from access to better defined hESC lines and ex vivo culture conditions. In order to realize this potential for hESCs we need to understand the molecular mechanisms governing self-renewal and pluripotency that guide the development of processes controlling the expansion and differentiation of stem cells. The proposed work will indirectly contribute to the design of diagnostics and therapies by identifying core renewal and differentiation regulatory networks and thus better defining of the functional capacities of hESCs. Diagnostics for therapy is one benefit, but diagnostics for protecting the health of Californians may be another benefit realized from the proposed work. California leads the nation and the world in establishing regulatory practices for environmental and workplace chemical exposures. Leukemias are a disease of the blood forming stem cells that can be induced by pro-oxidant chemical exposure. Defining at the protein level how stem cells respond to oxidative stress may identify early diagnostic markers (biomarkers) of disease or exposure. Early identification leads to early therapeutic intervention. Better yet, well defined biomarkers for exposure enable better environmental and workplace regulatory practices.
Progress Report: 
  • A central goal of our CIRM SEED proposal was to use innovative unbiased approaches to discover novel proteins that turn genes on or off in pluripotent stem cells. An understanding of what are these proteins that act as genetic switches and how they function is of significant importance to efforts to use pluripotent stem cells to model disease states in the lab or to provide a source of cells of therapeutic interest for transplantation. We have been successful in our efforts, in that we identified a novel protein that appears to play an unexpected role in the regulation of gene activity in pluripotent stem cells. In addition, we have identified another protein that is critical to maintain the DNA of pluripotent stem cells is a state accessible to other proteins. Our research is therefore providing an integrated picture of what are the genetic switches that turn genes on or off in pluripotent stem cells, what genes do they regulate, and how is their access to DNA regulated. Some of our results have recently been published, while other research is ongoing. In parallel, we have been very successful at transferring expertise to the biotechnology sector in California. In particular, two highly qualified lab members accepted senior scientist positions at top biotechnology firms in California (iPierian and Genentech).
  • A central goal of our CIRM SEED proposal was to use innovative approaches to discover genes that control human embryonic stem cells, with the idea that this knowledge may lead to improved methods for growth and/or differentiation of human pluripotent stem cells in a clinical setting. In the past year we have continued to make significant progress on these efforts. We have found a factor that acts to turn other genes on or off and is active in embryonic stem cells. We have put a considerable amount of effort into optimizing methods to identify exactly what genes this factor controls. Our results show that this factor directly regulates pluripotency-associated genes. This is remarkable, since this factor had not to date been implicated in the regulation of pluripotency. These results put us in a position to characterize the function of this factor in embryonic stem cells in greater detail. In addition, we are applying knowledge gained from our studies to develop methods to enhance the ease with which human pluripotent stem cells are propagated. Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, are notoriously more difficult to grow than their mouse counterparts, and this has significantly hampered the ability to use existing human pluripotent stem cells to model disease. We have developed conditions that facilitate the propagation of human pluripotent stem cells in a state that resembles mouse ES cells, where they are easier to propagate and grow more rapidly. These findings, while preliminary, suggest that we have the opportunity to explore a transition of human pluripotent stem cells to a state that is easier to culture and manipulate genetically. Thus, the CIRM SEED award has allowed us to discover a novel regulator of pluripotency genes, and to develop conditions that may lead to improved culture and manipulation of human pluripotent stem cells.

© 2013 California Institute for Regenerative Medicine