Abstract
Purpose :
Corneal epithelial stem cells (CESCs) and their progeny the TA cells play key roles in corneal epithelial homeostasis and proper wound healing. Both CESCs and early TA cells reside within the limbal epithelial basal layer, infrequently divide, and have extensive proliferative capacity. Early TA cells migrate into the corneal epithelium and mature into late TA cells, with diminished proliferative capacity. Late TA cells eventually differentiate into the upper layers of the corneal epithelium. This model has been proposed for decades; however, the molecular characteristics of the early and late TA cells remain unclear because of difficulties in obtaining discrete cell populations for analyses.
Methods :
We conducted scRNA-seq on isolated corneal and limbal cells using a 10X Gemonics single cell RNA seq pipeline to obtain discrete populations of these cells. Cell populations were distinguished by t-distributed stochastic neighbor embedding (tSNE) using a Loupe Cell Browser. To further define the subpopulations, Seurat analysis was conducted. Differential protein expression patterns were validated by immunofluorescence
Results :
We randomly profiled a total of 2,513 cells isolated from mouse corneal/limbal tissues. Unbiased clustering detected 10 distinct populations in corneal/limbal tissue, including 7 clusters of epithelial cells and 3 clusters of mesenchymal cells, which were identified by their unique molecular signatures. Among the epithelial cells, we identified 3 corneal/limbal epithelial cell subpopulations: Stem/early TA cells, late TA cells, and differentiated corneal epithelial cells. Each population displayed spatially distinct immunostaining patterns. For example, Growth arrest-specific 1 (Gas1) and suppressor of cytokine signaling 3 (socs3) were the most significant among 431 genes that represented the specific signature of early TA cells. Gas1 and socs3 proteins were preferentially detected in the mouse limbal epithelial basal layer, validating the robustness and reproducibility of our scRNA-seq data. We also identified PCNA clamp associated factor and thioredoxin interacting protein as novel regulators of the transition from early to late TA cells.
Conclusions :
Our data establish a comprehensive atlas of corneal/limbal epithelial cells, which form the foundation for further dissecting the roles of stem/TA cell populations in disorders of corneal epithelium.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.