ESC-B5 cells were incubated with or without the indicated concentrations of CTN for 24 h. (A) Cells were dissociated with trypsin–EDTA, and cultured in a medium without LIF to induce differentiation. EBs were formed with the hanging drop method, as described in the Materials and methods section. (B) ESC-B5 cells were incubated with or without the indicated concentrations of CTN for 24 h, and then treated further with 50 ng/ml NGF for 14 days. Cell extracts (60 μg) were immunoblotted with anti-MAP-2 antibodies. (C) Mouse blastocysts were treated with or without CTN (10–30 μM) for 24 h, and observed in culture for 72 h. Morphological assessment was used to identify blastocysts as hatched, ICM(+), ICM(++) and ICM(+++). The total numbers of blastocysts examined in each group were 260 (control), 272 (10 μM), 265 (20 μM) and 290 (30 μM). Results are means±S.D. **P<0.01, ***P<0.001 compared with the CTN-free group.