Hi all,
I'm analyzing Xenium data (brain tissue with the 5K Prime Mouse Brain panel). I followed the Seurat v5 pipeline (loadXenium(), removing cells with nFeature_Xenium < 10, and SCTransform normalization, PCA and Clustering). When I performed clustering, I found that some cell clusters expressed both neuronal and glial markers (and in some cases, endothelial cell markers as well). When I checked these cells in Xenium Explorer, they indeed contain transcripts from neurons, glia, and endothelial cells. I understand that with in situ data, we inevitably have some noise signal, but I'm unsure how to handle these clusters. Should I interpret them as mixed cell populations, or should I compare the expression levels of neuronal/glial/endothelial transcripts and annotate cells based on the most dominant cell type markers, while ignoring the other marker expressions?
Any suggestions would be appreciated!
Sincerely,
Song.
Is your experiment on wild type mouse or do you have a disease model ? Does the segmentation of the incriminated cells seems legit on Xenium explorer ? Which type of glial cells are you talking about ? What are the genes you believe are more specific to neurons or your glial cells ? Are your mixed marker cells located everywhere on your brain or is it region specific, like a brain lesion ?
I remember having these small clusters of cells in single cell experiments with both neuronal and oligo markers, which are often marked as doublets. In a disease context, it has been shown that microglia for example are eating up myelin residues and could very well contain oligos transcripts. Neurons and oligos are also tighly connected, it is highly probable that on some positions of the sectionning some neurons and oligos are touching or even overlapping. Moreover, a Xenium slide in 10µm thickness (if I am not mistaking), in this z dimension some oligos might have processes (with oligo transcripts) coming over a neuron body. The staining only capture cell body, so a transcript from an oligo process on top of a neuron would counted in that neuron body. For the endothelial cells, I would guess that with the same reasoning, if you are overlapping tiny blood vessel.