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2006;10:375C388. as a surrogate for normal stem cells and radiation-resistance CSCs respectively. The SCID mutation that underlies the immune deficiency in several mice strains has previously been shown to cause general defects in DNA repair [18]. We hypothesized that mice made up of the SCID mutation might be hypersensitive to radiation, thus limiting their power as recipients for the assessment of the radiation sensitivity of transplanted cells. To identify the optimal strain of mouse for our studies, i.e. with the lowest radiation-sensitivity, we analysed previous radiation exposure results from Epimedin A1 impartial and unrelated studies on the effects of irradiation on three strains of immunocompromised miceCtwo with the SCID mutation (NOD.SCID and NSG) and one without (NRG). Strains harbouring the SCID mutation exhibited increased mortality at doses less WASF1 than 50 Gy (Physique ?(Figure1A).1A). In comparison NRG mice, with immune deficiency mediated through Rag1 deletion rather SCID mutation, did not show increased mortality at doses of 65Gy or less. To further minimise the effects of radiation on the recipient and maximize the dose that could be delivered to the tumours, lead shielding was constructed to localize the irradiation (Physique ?(Figure1B).1B). To ensure the shielding was effective in reducing non-targeted radiation, and quantify the internal dose received, to the site of injection, thermo-luminescence dosimeter (TLD) chips were surgically implanted into the scrotum and small intestine of mice. The shielding reduced the radiation reaching the small intestine to 3.5C6.5% of the external dose while only partially reducing the radiation reaching the testes to 59C61.5% (Figure ?(Physique1C1C). Open in a separate window Physique 1 t-hESC derived tumours are radiation resistant compared with their normal counterpartA. Comparison of mouse survival for three staining of immunocompromised mice NOD/SCID, NOD/SCID Il2?/? (NSG) and NOD Rag1?/? Il2r?/? (NRG), used to quantify xenotransplantation post whole body irradiation illustrating relative tolerance to irradiation. Each collection represents a separate cohort of mice. Story explains radiation dose and the number of mice per cohort in parentheses. B. Overview of shielding apparatus for targeted irradiation highlighting irradiation chamber, lead shielding and restraint. C. Internal dose mice received while in shielding-apparatus measured using thermos-luminescence dosimeter (TLD) chips demonstrating effective shielding of body cavity by shielding apparatus (= 3). D. Schematic of experimental design to initiate, radiate and quantify testicular tumours. E. Outgrowth (teratocarcinoma from t-hESC or Epimedin A1 teratoma from hESC) volumes estimated via weekly palpation show no increase in those derived from hESC following either 10 or 20Gy doses gamma-radiation (each series represents 1 mouse). F. Final outgrowth (teratocarcinoma from t-hESC or teratoma from hESC) volume was quantified by displacement 4 weeks post irradiation show significantly larger tumours derived from t-hESC compared to hESC. G. Immuno-staining post-harvest recognized OCT4+ cells in both non-irradiated and 10Gy irradiated teratocarcinoma derived from t-hESCs. Colour deconvolution of the images allowed better visualization of OCT4-positive cells. By using this experimental configuration, the effect of irradiation on growth of teratocarcinomas derived from t-hESC and teratomas from hESC was quantified (Physique ?(Figure1D).1D). After intra-testicular injection with either hESC or t-hESC, mice were palpated weekly and irradiated when outgrowth volume reached 0.25C0.45 cm3. Estimation of volume by palpation showed Epimedin A1 that hESC derived teratomas ceased to continue growing after exposure to 10Gy and 20Gy irradiation and teratomas irradiated with 20Gy had significantly shrunk (paired- = 0.002) from their initial size. Teratocarcinomas nevertheless produced from t-hESC, did not stop to grow after contact with 10Gcon irradiation (Shape ?(Figure1E)1E) and with 20Gy irradiation didn’t display the significant decrease in size seen with hESC-derived teratomas. A month outgrowths were harvested and more precisely quantified by displacement later on. There is no factor in quantity between nonirradiated outgrowths produced from t-hESC and hESCs. Nevertheless, t-hESC derived teratocarcinomas significantly were.