Abstract
Purpose :
Schwann cells (SCs) ensheath axons in the peripheral nervous system and support the regeneration of axons after injury. As little is known about these resident corneal glial cells, we pursued single cell RNA-seq analysis to identify their transcriptomes. SC protein biomarkers were validated by immunostaining to illuminate the architecture of SCs in whole corneal tissue.
Methods :
The whole cornea (excluding the corneal-limbal tissue) from adult rabbit eyes (n=10, both sexes) was isolated. The single cell preparation was subjected to droplet-based scRNA-seq (10X Genomics) generating data on 7,555 individual cells. The entire procedure was replicated from a different batch of corneas generating data on another 10,057 individual cells. The gene expression matrix output from CellRanger (10X Genomics) of the aggregated data was subjected to unsupervised clustering and dimensionality reduction. Specifically, the 1500 most highly variable genes were used for neighborhood graph generation (using 20 nearest-neighbors) and dimensionality reduction with UMAP. For cross-species validation, antibody staining for the proteins representing highly expressed SC transcripts was done using mouse corneas and whole mount stained tissue was subjected to confocal microscopy.
Results :
The scRNA seq analysis of rabbit corneas produced 18 cell clusters, with the largest encompassing several for keratocytes, along with several for epithelial cells, as well as, distinct cell clusters for inflammatory cells, immune cells and Schwann cells. The corneal SC cell cluster revealed that Scn7A, Plp1, Gfra3, Sox10, and L1cam, including several other widely accepted mature SC markers, were highly expressed. The expression of the translated protein for these transcripts, as validated in mouse corneas, showed distinct staining around axon processes co-stained for β3-tubulin in the corneal stroma. Sox10 staining was distinctly nuclear in SCs. Finally, myelin basic protein (MBP) staining was limited to myelinated SCs localized at the limbus, confirming the absence of Mbp and several myelinating gene transcripts in our scRNA seq data derived from corneas lacking the myelinated limbal tissue.
Conclusions :
Our findings, for the first time, identify important molecular insights of corneal SCs. These findings will help to advance the study of corneal SCs by providing a much needed molecular toolbox for understanding SCs in corneal nerve repair and disease.
This is a 2020 ARVO Annual Meeting abstract.