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. 2021 Mar 23;11:6617. doi: 10.1038/s41598-021-84278-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Impaired dopaminergic differentiation in human pink1^-/- midbrain-specific organoids. (A) Representative bright field images of midbrain-specific organoids at different time points of differentiation. (B) Quantification of the size of the organoids by measuring their area in the images shown in (A). Arrow head indicates start of differentiation and arrow indicates start of maturation. Data points represent mean ± SEM. N = 3 independent generations of organoids, each starting with 48 organoids/line. (C) Representative images of day 30 midbrain-specific organoids sections (wt left, pink1-/- right) stained for Tuj1 (green) and TH (red). (D) Quantification of neuronal content of organoid sections measured as sum of Tuj1 positive pixels (neuronal marker) normalized by Hoechst positive pixels (nucleus marker) at day 0, 2, 4, 6, 14, 18, 22, 25, and 30 of differentiation. (E) Quantification of dopaminergic neuronal content measured as sum of Tuj1/TH double positive pixels normalized by Hoechst positive pixels. Data points represent mean ± SEM. P values are calculated by two-tailed Mann–Whitney-test. N = 3 independent generations of organoids, *p < 0.05, ***p < 0.001 ****p < 0.0001. (F) Heatmap displaying the features extracted from the image analysis and cluster analysis.