Fig. 4. Impaired dopaminergic and general neuronal differentiation in GBA-PD patient-specific midbrain organoids.

a Levels of extracellular dopamine in culture media at DIV60 were lower for GBA-PD organoids when compared with controls, measured by ELISA. The data represent a summary of five independent differentiation experiments for all cell lines. Wilcoxon T-test; ***p < 0.001. b Quantification of TH protein levels and representative western blot at DIV60 showing decreased levels of the protein in heterozygous GBA-N370S organoids. Data represents a summary of five independent differentiation experiments normalized to the mean of the controls per batch. Wilcoxon T-test; ***p < 0.001. c Representative images of DIV30 midbrain organoids sections stained for TH (red), MAP2 (green), nuclei (blue) (scale bar, 200 μm). d Immunofluorescence images of sections from Fig. 3c acquired at 40× (scale bar, 50 μm). e High-content automated image analysis of immunofluorescence stainings of dopaminergic neurons in organoids at DIV30 expressed as the proportion of cells expressing TH normalized by total nuclei. Data represents a summary of six independent differentiation experiments normalized to the mean of the controls per batch. Wilcoxon T-test; ***p < 0.001. f Neurite branching is less complex in dopaminergic neurons from GBA-PD organoids at DIV30 when compared with controls, measured by the number of nodes (branching points) and links (branches) extracted from the skeletonization of TH mask by the algorithm used for image analysis. Representative immunofluorescence images of TH+ neurons (yellow) showing less complex arborization in GBA-PD condition (scale bar, 50 μm) and graphic illustration of the morphometric features; links, nodes and skeleton. Data is normalized to the mean of the controls per experiment. n = 6, Wilcoxon T-test; *p < 0.05. g GeneGO MetaCore^TM enrichment analysis by process networks showing the top 20 overrepresented processes in DIV30 organoids. h Mean firing rate detected by individual electrodes of a multi-electrode array (MEA) system at DIV15 showing that mutant organoids are less electrophysiologically active. The data represent a summary of four independent differentiation experiments for all cell lines. Values are normalized to the mean of the controls per experiment. Wilcoxon T-test; ***p < 0.001. Upper panel shows a representative image of a midbrain organoid positioned on an 8-electrode array in a 96-well tissue culture plate (scale bar, 500 μm). i Decreased levels of TH+ cells normalized to the total neuronal population at DIV15 using the early neuronal marker TUJ1 and DIV30 using the late neuronal marker MAP2.