Towards a 3D spheroid system for modelling leukodystrophies 115 4 Single-cell RNA sequencing reveals cellular diversity in OL spheroids in all HD lines To further elucidate the cellular composition and developmental trajectory of the spheroids, we performed single-cell mRNA sequencing (scRNA-seq) on both HD and LD lines at days 100 and 150 of differentiation. Following quality control and data preprocessing, 27 distinct cell clusters were identified in organoids of HD lines (Fig. 2A). In addition, both clusters were present at both timepoints (Fig. 2B). We showed that there are no residual pluripotent cells and all cells show neuroectoderm identity (Supplementary Fig. 1A-D). The cells show gene expression profiles indicating neuronal cells of medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) (Supplementary Fig. 1G-L). Annotation of the clusters, using differentially expressed genes (Fig. 2C, Supplementary Fig. 1E-N) and predefined marker genes, revealed the presence of all three brain cell lineages, namely oligodendrocytes, astrocytes and neurons with both excitatory and inhibitory features (Supplemental Fig. 1E-M). In line with our expectations, microglia-like cells were not present in this model (Supplemental Fig. 1N). We present that HD spheroids provide cellular diversity as in the developing brain. LD lines map well onto HD data and show signs of disease specific changes Next, was to determine whether the current experimental set-up allows for the comparison between diseases. To start, we evaluated potential sources of experimental variability, including donor institute and batch effects. Principal component analysis (PCA) revealed that PC1, accounting for 34.1% of the variance, was not associated with these factors, as samples from the same institute and batch appeared distributed across PC1 (Fig. 3A). In contrast, PC2, explaining 16.7% of the variance, clearly separated samples by differentiation timepoint (D100 vs. D150). Notably, line E70 was the only sample positioned incorrectly on the y-axis, just above zero, and it clustered far from its isogenic control (Fig. 3A). Comparison of cell percentages per cluster showed that time significantly influenced cell proportions in 7 out of 27 clusters (Fig. 3B-D, Supplemental Fig. 2). Consistent with later-stage maturation, two of the three oligodendrocyte lineage clusters (OPC and cycling OPC) significantly increased over time (Fig. 3E-F).
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