Fig. 1

CHAT⁺ spinal MNs can be induced from iPSCs in 28 days using a previously published protocol. (A) Schematic diagram of the 28-day MN differentiation protocol established by Du et al⁸. (B) Representative morphology of cells during the course of differentiation from day 0 to day 28. (C) Representative staining of cells at each differentiation stage with stage-specific markers. OCT4/SOX2 staining for iPSCs on day 0, PAX6 and NESTIN staining for NSCs on day 6, OLIG2 staining for MNPs on day 12, ISL1 and MNX1 staining for early MNs on day 18 and CHAT staining for mature MNs on day 28 of differentiation. (D) The proportion of PAX6⁺ and NESTIN⁺ cells on day 6, OLIG2⁺ cells on day 12, ISL1⁺ and MNX1⁺ cells on day 18 and CHAT⁺ cells on day 28 of differentiation. N = 3 cell lines, n = 3 replicates. All representative images were from the iPSC line ALSH47C1. Scale bar, 100 µm. See also Fig. S1. (E) RT-PCR data showing the mRNA expression of PAX6 on day 6 (left up), OLIG2 on day 12 (right up), ISL1 and MNX1 on day 18 (left bottom) and CHAT on day 28 (right bottom) of differentiation compared to iPSCs on day 0. GAPDH was used as a house-keeping reference gene. Full-length gels are presented in Fig. S2.