Most knowledge of the effects of prenatal exposure to ionizing radiation (IR) is based on atomic bomb survivors and animal studies data. We are examining what effects CT exposure may have on the human embryo using hESC in culture as a model for studying the effects of IR on the hESC cellular biology. hESC have been proposed for therapy in replacing damaged cells in organs such as the brain and heart in patients who, through various disease processes, sustained the loss of vital differentiated cells. Numerous trials of stem cell therapy using hESC or induced pluripotent stem cells, are underway. Yet, no one fully understands the effects of even diagnostic CT ionizing radiation exposure on these cells while they are delivered in vivo for therapies of such patients. Thus, we examined hESC irradiated in situ in a tissue-equivalent, organ-torso, phantom using a commercial CT, at the diagnostic test settings routinely in use. We asked if such exposures induced cell responses such as changed growth, gene expression, or possibly induced mutations in the genomes. Finally, radiation safety concepts have been generally based upon the assumption that all of us, regardless of our unique genomes, are equally susceptible to the deterministic and stochastic effects of ionizing radiation, even at low doses. However, we today know about multiple genetic mutations, such as the BRCA-1 and BRCA-2 mutations, which affect DNA repair processes to the extent that a percentage of individuals with these genetic changes have an increased risk of developing certain cancers. When an individual has DNA repair deficiency, it stands to reason that exposure to any DNA-damaging levels of ionizing radiation is likely to be more detrimental than to an individual without such a deficiency. By using the multiple hESC lines available to us in the NIH, each genetically distinct, we began to examine differences between the various hESC lines responses to relevant, low-dose, ionizing radiation exposures. Using the increase in the hESC colony area as a surrogate measurement for cell proliferation, we assessed the effect of IR on seven lines of hESC. The immediate effect of hESC exposure to IR is some cell death by apoptosis. After an initial drop in the cell population, all lines show recovery of their growth after 24 to 48 hours. We found that relative cell survival (RCS) varied for hESC lines. The RCS of the most radiation sensitive H7 cell line was ca. 1.5 times less than that of the least sensitive WA24 cells. Interestingly, we found strong direct correlation between RCS and cell population doubling times; meaning that faster growing hESC are more prone to some apoptotic cell death after exposure to IR than slower growing cell lines. One possible explanation of this observation could be that the faster growing cell lines have higher percentage of cells in S phase. That could reflect a higher sensitivity to IR of cells in S phase; perhaps they cannot effectively repair their damaged DNA during replication, and thus more cells die from apoptosis. Results of our genomic mutation study of hESC suggest that exposure to a modest dose of IR (0.2Gy) did not result in any detectable increase in genetic alterations occurring within multiple cancer hotspot regions of the genome in four hESC lines. Nonetheless, a higher dose of IR (1Gy), that led to much more cell death within 24 hours after exposure, resulted in one detectable gene sequence variation in one of the four hESC lines tested. We believe that our first results support a need for further studies of genetic alterations in human cells caused by IR using next generation sequencing. It should be possible for us to sequence increasingly larger portions of the hESCs genomes with progressively deeper coverage. We plan to extend this particular work to whole-genome sequencing.
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