Abstract
We examined two biological endpoints in mouse neurosphere cells for evaluating the characteristics of stem cells; the repair kinetics of X-ray-induced DNA double strand breaks (DSBs) and the pathway toward immortalization by successive subculture of the cells. Fibroblast cells and neurosphere cells were obtained from embryos of ICR mice at day 14.5 of gestation. First, we examined the repair kinetics of DSBs after 1 Gy of X-irradiation by scoring phosphorylated histone H2AX (gamma-H2AX) foci and chromosome breaks visualized by premature chromosome condensation (PCC). The results indicated that the significantly less gamma-H2AX foci and PCC breaks were evident in the neurosphere cells than in the fibroblast cells until 1 h after irradiation, suggesting that the repair ability of DSBs in the neurosphere cells is more efficient than that in the fibroblast cells. Second, to establish a stable neurosphere cell line, we examined three successive subculture protocols whereby the neurosphere cells were plated at a density of 2X105 cells into a 25-cm2 flask and subcultured every 3 days (3T2), 5 days (5T2) or 10 days (10T2), with two additional trials (3T2 and 10T2) for the fibroblast cells as controls. We found that the neurosphere cells subcultured by the 5T2 and 10T2 protocols and the fibroblast cells subcultured by the 10T2 protocol extremely extended the lifespan (>100 PDN) whereas both cell strains subcultured by the 3T2 protocol senesced. Chromosome analysis revealed that chromosome numbers in the 50% lifespan extended neurosphere cells showed diploid whereas those in the fibroblast cells showed triploid and tetraploid. These results suggest that the pathway toward immortalization is different between the neurosphere cells and the fibroblast cells.
