CTX0E03 is a human neural stem cell line, which was derived from human somatic stem cells following genetic modification with c-mycERTAM, a conditional immortalizing gene. This transgene generates a fusion protein that stimulates cell proliferation in the presence of a synthetic drug 4-hydroxy-tamoxifen (4-OHT). The cell line is clonal, expands rapidly in culture (doubling time 50–60 h) and has a normal human karyotype (46 XY). In the absence of growth factors and 4-OHT, the cells undergo growth arrest and differentiate into neurons and astrocytes.
Cell lines were routinely cultured, for 1 h at 37°C, in tissue culture flasks freshly coated with mouse laminin (20 μg/ml in DMEM:F12). Growth medium supplemented with 4-OHT, was changed 3 times per week. When 70–90% confluent, cells were passaged using trypsin (0.25%) for 5–10 min at 37°C, followed by treatment with soybean trypsin inhibitor (0.25 mg/ml). Cells were spun down (800 × g for 5 min at room temperature) and resuspended at an appropriate density in full growth medium supplemented with 100 nM 4-OHT. Cells were then reseeded for expansion or experimental use.