Towards a 3D spheroid system for modelling leukodystrophies 125 4 Although we identified DEGs that were consistently regulated, GSEA for the oligodendrocyte lineage and other cell clusters (data not shown) is very variable by disease but also per cluster and timepoint. At this stage of the analysis, it is too early to make any conclusion based on the pathway analysis. Hence, we do not display it for the other lineages. Disease specific differential gene expression in astrocyte lineage Next, we investigated the astrocyte lineage, to be specific the glia progenitors, immature astrocytes and astrocytes. To obtain the most relevant findings, we visualized genes significant at both timepoints following the contrasts HD x GLD, HD x 4H or HD x CD and plotted the logfold2 change against each other. In the glia progenitors CSMD1, DPP6 and CHCHD2 are again significant and most drastically down regulated (Fig. 8A). In immature astrocytes and astrocytes they also occur but not always significant at both timepoints (Fig. 8B-C). Interestingly, in GLD there is quite a large cluster that is significantly upregulated in D100 and downregulated in D150. For 4H, SERPIN1, HOPX and CD9 are amongst the most upregulated genes in all timepoints and all three clusters (Fig. 8D-F). HSE4, NNAT, RALYL, DLGAP1 are consistently amongst the most down regulated genes in immature and mature astrocytes (Fig. 8E-F). For CD, there is a very large cluster of genes that is significant at both timepoints, but are upregulated in D100 and downregulated at D150. SPARCL1 is downregulated in both timepoints of glia progenitors and immature astrocytes, and only astrocytes on D150 (Fig. 8G-I). In conclusion, for GLD and CD the astrocyte lineage shows inconsistent up and downregulation of significant genes. Pointing towards drastic changes at the different timepoints, which might be interesting for future research. Additionally, for all LDs some significant genes for future research are identified.
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