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Fig. 3 | Acta Neuropathologica Communications

Fig. 3

From: Synapse alterations precede neuronal damage and storage pathology in a human cerebral organoid model of CLN3-juvenile neuronal ceroid lipofuscinosis

Fig. 3

A large fraction of CLN3Q352X hiPSC-derived cerebral organoids exhibit impaired growth. a CLN3Q352X organoids fail to expand and form neuroectodermal protrusions at three days of differentiation (3DOD) compared to the Control organoids. Scale bar, 200 μm. b Size measurements of organoids at 3DOD show a decrease in the area of the CLN3Q352X organoids. Area was calculated on ImageJ (NIH) by manually delineating the perimeter as regions of interest (ROI). Data points represent single organoids from three independent derivations (total n = 20 organoids). Comparison was performed applying a Mann-Whitney test between the two groups. c A proportion of CLN3Q352X organoids remains underdeveloped after 55DOD. Small Matrigel-embeded organoids are highlighted with a dashed yellow circle. d Quantification of the percentage of CLN3Q352X mutant organoids reaching a complete development. Data points represent percentage per organoid derivation (total n = 13 different organoid generations). Comparison between the two groups was performed with an unpaired t-test with Welch’s correction. For B and D, data is represented as mean ± SEM (p < 0.01**, p < 0.0001****). e Fully developed organoids from both Control and CLN3Q352X mutant genotypes express forebrain marker FOXG1 (green) and cortical layer identities: SATB2 (red) for later-born superficial-layer identity and CTIP2 and TBR1 for early-born deep-layer identity (CTIP2 and TBR1, both green), at 55DOD. They also express the neuronal marker MAP 2 (red) and neural stem cell markers SOX2 (white) and Nestin (green). Scale bars, 200 μm. f. Representative western blots for the some of the markers mentioned above. Quantifications show similar expression in fully developed CLN3Q352X organoids compared to the Controls at 55DOD. Data points represent single different organoids (n = 7 different organoids per condition). g Representative TEM pictures of the ultrastructural neuronal features of both organoid cultures at the same stage. Nerve fibers are highlighted in a yellow dashed boxed and higher magnification picture is offered at the right side to visualize neurofilaments (nf; <, both in red), which are present in both genotypes, as well as synaptic vesicles (sv; black dashed circles). Scale bars represent 1 μm, 200 nm for magnified areas

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