Chapter 4 112 astrocytes, are present in our model. Next, we performed single-cell mRNA sequencing (scRNA-seq) to further elucidate the cellular composition of the spheroids. Cluster annotation showed diverse cellular populations. Some clusters were significantly different over time, emphasizing the dynamic nature of the models. Interestingly, the two clusters cycling radial glia and neural progenitors were severely underrepresented in leukodystrophy organoids. The scRNA-seq data was subsequently used to investigate the validity of comparing all healthy donors (HD) against specific LDs, by comparing the findings of isogenic comparisons and HD x GLD. We showed that the most significant genes are consistently up or downregulated in both isogenic comparison and HD x GLD. Next, the scRNA-seq data was used to investigate which specific cell clusters highly express the genes GALC, POLR3A and ASPA, which are causative in three leukodystrophies respectively, Globoid Leukodystrophy (GLD), 4H Leukodystrophy, and Canavan Disease (CD). As no particular cell cluster seemed to be more relevant in all leukodystrophies, but all leukodystrophies show myelin pathology in the clinic, we decided to investigate the DEG of all three clusters of the oligodendrocyte lineage as well as gene set enrichment analysis (GSEA). Interestingly, we identified many DEGs but only a few could be directly linked to oligodendrocytes, such as down regulation of MAG (Myelin Associated Glycoprotein) in 4H. GSEA showed possible relevance of translation and oxidative phosphorylation in LD pathology, as they are often significant, but further research is required to confirm their involvement. DEG analysis on a selection of clusters from the neuron and astrocyte lineage again showed plenty differential genes, but their relevance is yet to be determined. Together, this work shows that 3D spheroid models are a valuable platform for studying the cellular and molecular mechanisms underlying leukodystrophies and guiding therapeutic development.
RkJQdWJsaXNoZXIy MTk4NDMw