Fig. 5.

Replacement of small molecule compounds with more specific and less toxic inhibitors. a Morphology of EBs, which were derived from 201B7, treated with vehicle (DMSO), DS, DSB, or DSC on day 4. Massive cell death was observed after the treatment of DSB. Scale bar = 200 μm. Control, 0.09 % DMSO. DS, dorsomorphin and SB431542. DSB, dorsomorphin, SB431542, and BIO. DSC, dorsomorphin, SB431542, and CHIR99021. b The number of cells harvested from DSB- and DSC-treated EBs on day 14 per 100-mm culture dish of starting hiPSCs. n = 4, mean ± SEM. *, p < 0.05 (Student’s t test). c The number of cells harvested from DSC- and LSC-treated EBs on day 14 per 100-mm culture dish of starting hiPSCs. n = 4, mean ± SEM. LSC, LDN193189, SB431542, and CHIR99021. d Immunocytochemical analysis of motor neurons derived from DSC- or LSC-treated EBs for HB9, ISL-1 and βIII-Tubulin on day 7 of monolayer differentiation. Scale bar, 100 μm. d Quantitative analysis of the number of the cells positive for HB9 and ISL-1. n = 4, mean ± SEM. e Time-course analysis of the expression of PAX6, SOX1, NGN2, OLIG2, NKX2.2, HB9, and ISL-1 in EBs and during monolayer differentiation of motor neurons (MN) via quantitative RT-PCR. n = 4, mean ± SEM