The application of flow cytometry to examine damage clearance in stem cells from whole-body irradiated mice

Sarah A. Krueger*, Michele McGonagle, 1Olga Kovalchuk, Alvaro Martinez, George D. Wilson and Brian Marples**

Radiation Biology Group, Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan, U.S. and 1The University of Lethbridge, Alberta, Canada
* Presenter
** Primary Investigator

Stem cells are defined by their ability to produce pools of precursor and differentiated functional cells and as such, are widely regarded as the most critical target of radiation-induced carcinogenesis. Of particular interest are a small proportion of all stem cells referred to as side population cells (SP cells) that have been shown to demonstrate a universal stem cell phenotype. These cells can be isolated by flow cytometry based upon their ability to rapidly efflux Hoechst 33342 dye and typically only comprise 0.1% of the total bone marrow. Because of the pluripotent nature of these cells, the characterization of their radiation response after low-dose exposure may be significant in understanding long-term low-dose radiation risks. The aims of this project are to determine whether radiation-induced damage persists in SP stem cell populations after whole body low-dose radiation exposure and to characterize how immunological stress [treatment with bacterial lipopolysaccharide (LPS)] affects damage processing in SP cells.

The hypotheses being tested in this study are:

1. DNA damage persists in bone marrow stem cell populations after low-dose radiation exposures leading to genomic instability because low-levels of radiation-induced DNA damage are able to evade cellular damage recognition processes.
2. Immunological stress prior to radiation exposure enhances DNA damage processing in stem cell populations producing a radioprotective effect.

These hypotheses are being tested using a C57/Bl/6 mouse model after whole-body X-irradiation (0.01 Gy, 0.1 Gy, 1 Gy or 10×0.01 Gy fractions). The study is designed to examine three endpoints in the SP stem cells after radiation exposure: DNA double-strand break repair, cell death, and genomic instability. DNA double-strand break repair is assayed by scoring the residual γH2AX foci; cell death is measured using an apoptosis assay (Caspase-3/7 activation); and genomic instability will be evaluated by examining DNA methylation one month after irradiation. The effect of immunological stress as a modulator of in vivo radiation damage processing in stem cells will also be examined by treating the mice with LPS prior to irradiation and examining the same endpoints.

A mouse phantom was used to define our irradiation system in order to maximize uniform dose depth distribution and dosimetry for unrestrained whole-body irradiations. For initial studies, bone marrow cells were isolated from the femurs and tibias of 7-10 week old C57/Bl/6 mice thirty minutes post irradiation (or mock) giving an average yield of 4.0 x 107–10 x107 cells per mouse, with viability ranging from 85%–95% as determined by trypan blue exclusion. Following the established protocol of the Goddell Lab, bone marrow cells were stained with 5 μM/mL

DE-FG02-07ER64337 Hoechst-33342 for 90 minutes at 37°C and a sample of the bone marrow was treated with 100 μM/mL Verapamil, which blocks the efflux pump proteins. The stained cells were then resuspended in 4°C HBSS containing 2 μg/ml propidium iodide and the SP stem cells were sorted by flow cytometry. The samples were run on a BD Biosciences FACS Aria cell sorter equipped with a custom UV laser set up to detect both Hoechst Red and Hoechst Blue and SP cells were sorted either into 5 mL tubes, 1.2 mL tubes or 96-well plates to test the outcome assays. To date we have been successful at routinely staining, identifying and sorting the SP stem cell population and testing the sorted cells for DNA double-strand breaks by staining for γH2AX foci and examining cell death by looking at Caspase-3/7 activation. Analysis of the sorted cell populations has shown they demonstrate the expected surface markers, including positivity for stem cell antigen-1 (Sca-1). Proper isolation of the appropriate population is also confirmed by the absence of the SP cells after treatment with verapamil, as this reagent blocks the Hoechst efflux in the SP cell population.

The successful utilization of the endpoint assays in conjunction with the sorting of the SP population means that we are now in a position to begin the bulk of the experimental work. Once the first phase of studies has been completed in un-stressed mice, the entire protocol will be repeated with the addition of immunological stress (LPS treatment). We anticipate that the addition of immunological stress will protect cells from damage caused by low-dose radiation exposure and that the data as a whole will aid the definition of low-dose radiation risk in a dose range were current epidemiological data are lacking.

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