We studied the effect of ionizing radiation (IR) on continuous growth of seven hESC lines. Cells were exposed to 0, 0.2, or 1 Gy of X-rays, and the growth rates of cell populations were assessed by measuring areas of the same individual colonies versus time. The population doubling times (DT) of sham-irradiated cells varied from 18.9 to 28.7 hours for different cell lines. All cell lines showed similar reaction to IR, i.e. cell populations dropped within 24–48 hours post IR; after that they recovered and grew with the same rate as the sham-irradiated cells. The relative cell survival (RCS), i.e. the ratio of normalized cell population in the irradiated samples to that of the sham-irradiated ones varied from 0.6 to 0.8 after 0.2 Gy, and from 0.1 to 0.2 after 1 Gy IR for different cell lines. We found that the RCS values of hESC lines correlated directly with their DT, i.e. the faster cells grow the more radiosensitive they are. We also found that DT and RCS values of individual colonies varied significantly within all hESC lines. We believe that the method developed herein can be useful for assessing other cytotoxic insults on cultures of hESC.

We studied the effect of ionizing radiation (IR) on continuous growth of seven hESC lines. Cells were exposed to 0, 0.2, or 1 Gy of X-rays, and the growth rates of cell populations were assessed by measuring areas of the same individual colonies versus time. The population doubling times (DT) of sham-irradiated cells varied from 18.9 to 28.7 hours for different cell lines. All cell lines showed similar reaction to IR, i.e. cell populations dropped within 24-48 hours post IR; after that they recovered and grew with the same rate as the sham-irradiated cells. The relative cell survival (RCS), i.e. the ratio of normalized cell population in the irradiated samples to that of the sham-irradiated ones varied from 0.6 to 0.8 after 0.2 Gy, and from 0.1 to 0.2 after 1 Gy IR for different cell lines. We found that the RCS values of hESC lines correlated directly with their DT, i.e. the faster cells grow the more radiosensitive they are. We also found that DT and RCS values of individual colonies varied significantly within all hESC lines. We believe that the method developed herein can be useful for assessing other cytotoxic insults on cultures of hESC.Human embryonic stem cells (hESC) are unique models for studying genotoxic stresses including those produced by ionizing radiation (IR) because of their virtually unlimited proliferation potential 1,2 . In addition, their ability to differentiate into various tissues allows for studying the effects of IR on the developmental processes 3 . Studies of the effects of IR on hESC are also important because of the prospects of using these cells in regenerative medicine, which would require their transplantation or the transplantation of their differentiated products, and the imaging of the transplants with various techniques that may, like positron emission tomography (PET), utilize ionizing radiation 4 . During the past decade considerable knowledge was gained regarding the effects of IR on hESC 5 . It was shown that hESC are very sensitive to IR; the exposure to a relatively low dose of 1 Gy results in death of almost 30% of the cells 6 . The immediate effect of IR exposure of these cells is apoptosis; however, the surviving cells retain their pluripotency markers and ability to form all three embryonic germ layers as was evidenced by teratoma formation assay [6][7][8] . At the same time hESC have enhanced capacities to repair DNA damage as compared to differentiated cells 9 . It was found that these cells lack the G1/S checkpoint and stop their cell cycle progression after IR exposure by G2/M arrest instead [10][11][12] . Repair of DNA double strand breaks in hESC was shown to rely largely on homologous recombination [13][14][15] . A distinct feature of hESC is that in culture they propagate in tightly associated colonies 16 . When dissociated to a single cell suspension and plated back to a culture dish the majority of the cells die. Although treatments with certain factors can increase the plating efficiency of hESC 17,18 , proliferation in a colony is a normal physiological...