Fig 2: Time course of MN-specific markers expression(A) Immunofluorescence for Tubulin ßIII or HB9 in green fluorescence and of ISL1, SV2, ChAT and vAChT in red fluorescence in undifferentiated cells (day 0) and after 24 and 50 days of culture, as indicated. The nuclei were counterstained with DAPI (blue). Original magnification: 20x at day 0, 24 and 50, scale bar 100 µm; the last column shows details at bigger magnification (60x, scale bar 40 µm) of the day 50 staining. Images are representative of 5 independent experiments White arrows indicate the peri-nuclear localization of ChAT, yellow arrowheads indicate the dotted pattern of vAChT or SV2 that mark the synaptic vesicles.(B) ScatterPlot of the Day 50 colocalization between blue (nuclei) and green or red signal calculated using the Pearson’s coefficient, as indicated.

Fig 3: Immunofluorescence analysis of MN-specific markers(A) HB9 (green fluorescence) in undifferentiated cells (day 0) and after 24 days of culture. The nuclei were counterstained with DAPI (blue). White arrows indicate the nuclear localization of HB9 in the differentiated cells. Original magnification: 40x, scale bar 20 µm. Images are representative of 3 independent experiments.(B) HB9 fluorescence intensity graph; data are expressed as Mean ± SD (n = 3, ***p<0.001).(C) ScatterPlot of the colocalization between blue (nuclei) and green (HB9) signal calculated using the Pearson’s coefficient (r= 0.74).(D) immunofluorescence for vAChT (red) and NF-L (green) in undifferentiated cells (day 0) and after 24 days of culture. The nuclei were counterstained with DAPI (blue). Original magnification: 40x, scale bar 20 µm. Red rectangle represents an enlarged area: magnification 60x, scale bar 10 µm. Images are representative of 3 independent experiments.The graphs show the Mean ± SD of 3 diverse, randomly selected fields of 3 independent experiments. ***p<0.001.Scatterplot of the colocalization between blue (nuclei) and red (vAChT) signal calculated by Pearson’s coefficient (r= -0.11).