Figure 1

Experimental scheme of clonal mutation analysis of mouse GS and mGS cell lines. GS cells were established from neonatal testicular cells of male mice using MEF cells as a feeder cell layer in the presence of GDNF and FGF2. mGS cells were isolated from GS cell cultures by picking up cell colonies that changed morphology to a pluripotent stem cell-like appearance. mGS cells were maintained on MEF cells under a conventional mouse ES cell culture condition supplemented with leukemia inhibitory factor (LIF) and fetal bovine serum. Clonal parental cultures were derived from single cells of GS and mGS cell lines by plating each cell type at low cell densities and picking up clonally derived colonies, followed by deposition to 96-well plates. These clonal parental cultures were then expanded and maintained for a defined period (100 population doublings) and used for WGS and karyotype analyses. From each clonal parental culture, three subclonal cultures were further derived by recloning and then used for WGS. By comparing WGS data of parental and each subclonal cultures, de novo mutations that accumulated during 100 population doublings were analyzed. Two experimental sets (exp1 and exp2) of these clonal mutation analyses were carried out for GS and mGS cell lines, respectively. GS cell lines were also maintained under a standard bulk culture condition for up to 5 years (5, 36, and 60 months). Mutation accumulation in these standard GS cell cultures was analyzed by comparing WGS data obtained at 5, 36, and 60 months.