Spinal MN differentiation was performed based on methods described in previous reports (Klim et al., 2019 (link); Motosugi et al., 2021 (link)). Briefly, the culture medium for confluent hPSCs was replaced with an MN induction medium. The differentiation medium contained ½ Neurobasal (Thermo Fisher Scientific) and ½ DMEM-F12 (Thermo Fisher Scientific) supplemented with Gibco B-27 supplement (×1; Thermo Fisher Scientific), Gibco N-2 supplement (×1; Thermo Fisher Scientific), Gibco GlutaMAX (×1; Thermo Fisher Scientific), and 100 μM non-essential amino acids. The time point at which the medium was changed was defined as day 0 of MN differentiation. We used the following small molecules: 10 μM SB431542 (StemCell Technologies), 1 μM retinoic acid (StemCell Technologies), 100 nM LDN-193189 (StemCell Technologies), and 1 μM Smoothened agonist (StemCell Technologies) on days 0–5; and 5 μM DAPT (StemCell Technologies), 4 μM SU-5402 (StemCell Technologies), 1 μM retinoic acid (StemCell Technologies), and 1 μM Smoothened agonist (StemCell Technologies) on days 6–14. On days 10 or 14, the differentiation efficiency of MNs was analyzed using immuno-FISH analysis. All cells, except naïve cells, were cultured at 37°C in the presence of 5% CO2.
Motosugi N., Sugiyama A., Okada C., Otomo A., Umezawa A., Akutsu H., Hadano S, & Fukuda A. (2022). De-erosion of X chromosome dosage compensation by the editing of XIST regulatory regions restores the differentiation potential in hPSCs. Cell Reports Methods, 2(12), 100352.
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