The Response of Embryonic Stem Cells to Oxidative Stress
Researcher 1 - Name : Dr Steven Gieseg
Researcher 1 - Institution : University of Canterbury
RESEARCHERS:
Researcher 2 - Tina Yand- PhD Student
Researcher 2 - Institution : University of Canterbury
Period of funding: 12 months
Amount Funded: 32,770
Photo: Dr Steven Gieseg in the Laboratory
Free Radical Biochemistry Laboratory, School of Biological Sciences, University of Canterbury
The Response of Embryonic Stem Cells to Oxidative Stress.
Dr Steven Gieseg, BSc(Hons), PhD (Otago).
Stem cells are supposedly immortal cells, yet their gradual loss from the body is a fundamental part of the aging process. Surprisingly little is known about the death of stem cells or what kills them.
One of the major stresses all cells are subjected to is the damaging effects of oxidants and free radicals. Our research will look at how stem cells change or die when exposed to free radical stress. We intend to identify which natural antioxidants are present in stems cells protecting them and what level of damage they recover from. My Free Radical Biochemistry Laboratory at the University of Canterbury’s School of Biological Sciences has for the last decade examined how a type of white blood cell called macrophages, react to toxins and free radicals. It is the death of these macrophages within arteries that course heart disease and strokes. At the invitation of the Australian Stem Cell centre we have now branched out to examine these process in Human Embryonic Stem Cells (hESC).
Using techniques developed at the Australian Stem cell centre in Monash, Melbourne we will established and grow the hESC MEL-1 cell line before examining how these cells response to oxidative free radicals. This research will require considerable method development to find ways to expose the cells to oxidants within a environment which the cells can survive in. The Mel-1 line was developed in Australia by the Australian stem cell centre under full ethics approval and consent of the donors. This line has now been gifted to science for all to use for research.
This research has also the added advantage of bringing this exciting and extremely difficult technology to New Zealand and particularly the Canterbury region. To get this research up and running we were heavy involved in much of the debate and legislation development which result in the new ethics regulations governing the use of embryo derived stem cells. This laboratory was actual the first to get permission to import and use hESC for research.
Objectives of the Project
1) Characterise embryonic stem cell viability loss during oxidant exposure.
2) Determine whether stem cells undergo apoptosis or necrosis.
3) Determine the role of glutathione in protecting stem cells from oxidative stress.
Experimental Program
The below experimental program will be conducted over the seven months of the project. All procedures describe have been previously used in this laboratory for the study of monocyte cell lines and human macrophages.
1) Characterise stem cell viability during oxidant exposure.
The stem cells will be exposed to increasing concentrations of either hydrogen peroxide or the peroxyl radical generator AAPH to determine which concentrations trigger cell death. Cell death will be measured by MTT reduction and trypan blue exclusion assays.
2) Determine whether stem cells under go apoptosis or necrosis
Stem cells exposed to either hydrogen peroxide or peroxyl radicals will be examined for the activation of the effector caspases 3 and 6 using a fluorescence based assay23. Phosphatidylserine (PS) exposure on the outer cell membrane is a hall mark of apoptosis. PS exposure will be measured by flow cytometry analysis of stem cells stained with both propidium iodide and annexin-FITC.
3) Determine the role of glutathione in protecting stem cells from oxidative stress.
Intracellular glutathione levels will be measured by labelling the glutathione with the reagent monobromobimane and detecting the fluorescent derivative in cell lysates by high performance liquid chromatography. The loss of glutathione, possible caspase activation, PS exposure and loss of cell viability will be measured over the time of the incubations with the oxidants. This will provide considerable understanding of the mechanisms involved in embryonic stem cell death.
