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.
SYNOPSIS: The Principal Investigator (PI) hypothesizes that identification of chemical and oxidative response pathways will facilitate the optimization of human embryonic stem cell (hESC) culture and differentiation procedures, and proposes to apply comparative proteomics to characterize the oxidative stress response of hESCs and their progeny at the protein level using quantitative mass spectrometry (MS). Incorporating labeled internal standard techniques with isotope-coded affinity tagging (ICAT) technologies will enable quantitation of target peptides. Specifically, the PI proposes to: (1) Profile microsomal and cytosolic proteins in hESCs with an emphasis on quantitative analysis of oxidative response proteins. (2) Quantify the oxidative response proteins in committed hematopoeitic progenitors derived from hESCs. (3) Identify changes to the proteomic profile of hESCs and hematopoietic progenitors grown at 2% vs 20% oxygen and following chemically-induced oxidative insult. INNOVATION & SIGNIFICANCE: Aim 1 is to use MS technology to identify all cytosolic and microsomal proteins in ES cells. In Aims 2 and 3 the applicant will use ICAT technology to compare the abundance of all cytosolic and microsomal proteins in ES cells growing in 2 vs 20% oxygen. Similar experiments will be done on ES cells differentiated into specific hematopoetic lineages. Unclear significance and no innovation STRENGTHS: None WEAKNESSES: This is an entirely descriptive project with no attempt made to relate the anticipated results to ESC biology. One of the reviewers could not understand what hypothesis is being tested, nor how the applicant intends to use any of the massive amounts of data he/she would generate from these experiments. The applicants have very limited prior experience with global proteomics, and even more problematic is their lack of experience specifically with the ICAT technologies. The recruited expert in mass spec technology is a postdoc who has one prior publication in this area (middle author) and no experience in ICAT technology. DISCUSSION: One reviewer added that although the approach to proteomics was competent there were issues and results are not expected as described in the proposal. No further discussion following reviewers' comments.