Fig. 2: Differentiation into ventral midbrain progenitors and mesDA neurons.

a Schematic diagram of the DIV16 midbrain differentiation protocol using different concentrations of GSK3i ranging from 0.6 µM to 0.8 µM. Flow cytometry analysis of the percentage of (b) FOXA2 + , LMX1A + , OTX2+ and EN1+ cells, and (c) FOXA2 + LMX1A + , OTX2 + EN1 + , FOXA2 + LMX1A + OTX2 + EN1+ cells among H9 and 4X at DIV16 after administration of GSK3i at concentrations ranging from 0.6 µM to 0.8 µM. The data are presented as the mean ± SD; n = 5, from independent experiments. Two-way ANOVA followed by Sidak’s multiple comparisons test. d Representative immunofluorescence analysis of FOXA2, LMX1A, OTX2, and EN1 expression in H9 and 4X cells treated with GSK3i at concentrations of 0.6 µM, 0.7 µM and 0.8 µM. Scale bars, 50 µm. e, f Flow cytometry analysis of FOXA2 + LMX1A + , OTX2 + EN1 + , FOXA2 + LMX1A + OTX2 + EN1+ cells among H1, 4X-H1-NC1, GBA, 4X-GBA-C7, and 4X-GBA-C8 cells at DIV16 when GSK3i was 0.8 µM. The data are presented as the mean ± SD; (H1, 4X-H1-NC1, 4X-GBA-C8: n = 3; GBA, 4X-GBA-C7: n = 4) from independent experiments. An unpaired two-tailed t test was used to compare groups in (e) and one way ANOVA with Dunnett’s test for (f). g Schematic diagram of the DIV30 midbrain differentiation protocol. h Quantification of TH/DAPI, (FOXA2 + TH + )/TH, (EN1 + TH + )/TH-percentage cells among H9, 4X, at DIV30 when GSK3i were 0.7 µM and 0.8 µM, and H1 and 4X-H1-NC1 at DIV30 when GSK3i was 0.8 µM. The data are presented as the mean ± SD; (n = 3 all conditions excect n = 4 for 4X when GSK3i was 0.8 µM), from independent experiments. Two-way ANOVA followed by Sidak’s multiple comparisons test was used for H9 and 4X. An unpaired two-tailed t test was used to compare H1 and 4X-H1-NC1 groups. i Representative images for FOXA2, TH, EN1 staining at DIV30. Scale bars: 50 µm. Source data are provided as a Source Data file.