Corneal nonmyelinating Schwann cells illuminated by single‐cell transcriptomics and visualized by protein biomarkers

Abstract

The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal Schwann cells (nm‐cSCs). This anatomical organization is believed to be important for central vision. Here we employed single‐cell RNA sequencing (scRNA‐seq), microscopy, and transgenics to characterize these nm‐cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA‐seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm‐cSCs. To confirm protein expression of cSC genes, we performed cross‐species validation, employing corneal whole‐mount immunostaining with confocal microscopy in mouse corneas. The expression of several representative proteins of nm‐cSCs were validated. As the proteolipid protein 1 (PLP1) gene was also expressed in nm‐cSCs, we explored the Plp1‐eGFP transgenic reporter mouse line to visualize cSCs. Specific and efficient eGFP expression was observed in cSCs in adult mice of different ages. Of several putative cornea‐specific SC genes identified, Dickkopf‐related protein 1 was shown to be present in nm‐cSCs. Taken together, our findings, for the first time, identify important insights and tools toward the study nm‐cSCs in isolated tissue and adult animals. We expect that our results will advance the future study of nm‐cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.

The transcriptional profile of keloidal Schwann cells

“…3a ) were identified using well-established Schwann cell markers (Fig. 3b ) 17 , 27 . This analysis identified myelinating Schwann cells (SC-Myel), nonmyelinating Schwann cells (SC-Nonmyel), proliferating Schwann cells (SC-Prolif), Schwann cells additionally expressing genes typical for endothelial cells (SC-EC) or fibroblasts (SC-FB) and the previously described repair-like, profibrotic keloidal Schwann cells (SC-Keloid) (Fig.…”

“…Subset clusters expressing marker genes typical for melanocytes were removed. Schwann cells were characterized by the expression of established marker genes 17 , 27 . Differentially expressed genes were defined as genes displaying an average fold change >2.…”

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

“…A similar approach was applied to the ocular anterior segment in mouse, leading to identification of a novel marker for stem and early transit amplifying cells, TXNIP [ 14 ]. While this work was under review, two studies reported the single cell transcriptomic analysis of human limbal basal epithelium [ 15 ] and murine corneal non-myelinating Schwann cells [ 16 ]. Our study complements and expands these published data by providing a comprehensive single cell atlas of the whole developing and adult human cornea and adjacent conjunctiva that defines their development, limbal progenitor cells and their interactions with immune cells.…”

The transcriptional profile of keloidal Schwann cells

“…As the main goals of our study were the in-depth analysis of Schwann cells in the different scar types and their comparability across different single cell data sets, we next recalculated the combined integrated Schwann cell cluster, removed all melanocytes and performed subclustering (Figure 2). The different Schwann cell subclusters (Figure 2a) were identified using well-established Schwann cell markers (Figure 2b) 31,41 . This analysis identified myelinating Schwann cells (SC-Myel), nonmyelinating Schwann cells (SC-Nonmyel), proliferating Schwann cells (SC-Prolif), Schwann cells additionally expressing genes typical for endothelial cells (SC-EC) or fibroblasts (SC-FB) and the previously described repair-like, pro-fibrotic keloidal Schwann cells (SC-Keloid) (Figure 2a and b).…”

“…Subset clusters expressing marker genes typical for melanocytes were removed. Schwann cells were characterized by the expression of established marker genes 31,41 . Differentially expressed genes were defined as genes displaying an average fold change > 2.…”