Figure 2.

RA induces MNs from hESCs and iPSCs in the absence of extrinsic SHH activation. A, Early exposure to RA efficiently derives MNs from hPSCs with comparable yields and timing in the presence and absence of Pur. B, Patterning with RA achieves highest MN yields when initiated at a very early neural stage (day 1). Data are shown as mean ± SEM; n = 3 independent experiments; ***p < 0.001; **p < 0.01; *p < 0.05 versus RA initiated at day 1, Dunnett test. C, Live GFP expression in RA-patterned culture at day 16. Scale bar, 10 μm. Immunocytochemistry at day 7 for PAX6 (D), at day 16 for HB9, ISL1, and LIM3 (E), and at day 21 for NKX6.1 (green) and OLIG2 (red; F) confirms MN identity of RA-derived cultures. G, Quantification of markers presented in E and F. Immunocytochemistry at day 23 for HB9, ISL1, and TUJ1 (H) shows MNs at a more mature stage. I–K, Immunocytochemistry at day 16 for ISL1 (red) and HB9 (green; left), NKX6.1 (green) and TUJ1 (red; middle), and at day 23 for SMI32 (green) and ISL1 (red; right) for MNs derived from control (11A and 18B) and ALS patient-derived (27B) iPSCs via patterning with RA (J) or RA + Pur (K). I, Quantification of markers in J and K demonstrating similar, efficient propensity to generate MNs across multiple lines regardless of disease status. Scale bars, 100 μm unless labeled otherwise.