Chapter 4 122 Identifying clusters for follow-up analysis by expression of disease associated genes As we identified 27 clusters, we opted to focus on a subset of disease relevant clusters. Considering that the 3 diseases under investigation are all leukodystrophies, we are particularly interested in investigating the oligodendrocyte lineage. However, to investigating whether there are other cell clusters in OL spheroids that warrant our investigation, we investigated expression of key disease-related genes and downstream products linked to GLD, 4H and CD. Spatial expression analysis using UMAP revealed that GALC was primarily localized to the left side of the UMAP, encompassing astrocyte and glial progenitor clusters (Fig. 6A). In contrast, 4H related gene POLR3A displayed broad, nonspecific expression across multiple cell types without a distinct spatial pattern (Fig. 6B). Attempts to refine POLR3A localization by examining co-expression with POLR3B yielded no additional specificity (Fig. 6C). ARX, a gene previously implicated in 4H, was notably expressed in cycling cells, cycling radial glia, NPCs, excitatory neuronal progenitor 4 (NP4), inhibitory neurons, GABAergic neurons, SST neurons, and SST neuronal progenitors (Fig. 6D). While ASPA, the gene linked to CD, was only moderately expressed in astrocytes, its closely related gene NAT8L showed high expression in this NPC cluster as well (Fig. 6E-F). Interestingly, we found that GALC and POLR3A, associated with GLD and 4H respectively, were highly expressed in a subset of cells within the neural progenitor cell (NPC) cluster (Fig. 6A-B). To summarize, we will have a more detailed look at the oligodendrocyte lineage. Additionally, we decided that astrocyte lineage is particularly interesting to investigate regarding disease-gene expression in GLD and CD, we opted for glia progenitors, immature astrocytes and astrocytes. Additionally, previous research on 4H shows that the neuronal lineage requires attention, hence we focussed on some neuron specific clusters. To get a complete overview we decided to investigate, NPCs, excitatory NP1, 5HT neurons, inhibitory NP1, SST neurons and GABAergic neurons.
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