Abstract
Purpose :
In addition to stem cells (SC), the transit-amplifying cells (TAC) play an important role in tissue homeostasis, development, and regeneration. This study was to characterize the gene expression profile and signature genes of a TAC population in basal epithelial layer of human corneal limbus using single cell transcriptomics with a variety of culture models of human corneal epithelial cells (HCECs).
Methods :
Single cells isolated from the basal corneal limbus were subjected to scRNA-Seq processing using the 10x Genomics platform. Cell types were clustered by the graph-based visualization methods and an unbiased computational analysis. BrdU proliferation assays, immunofluorescent staining and real-time RT-qPCR were performed using multiple culture models of primary HLECs to characterize the TAC population.
Results :
Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters. A unique cluster (3.21% of total cells) was identified as the TAC entity based on its less-differentiated progenitor status and enriched exclusive proliferation marker genes with 98.1% cells in S and G2/M phases. The cell cycle-dependent genes were uncovered to be largely enriched by TAC population. A group of top genes were characterized morphologically and functionally at protein and mRNA levels. The specific expression patterns of RRM2, TK1, CENPF, NUSAP1, UBE2C and CDC20 were well correlated at time-/cycle-dependent manner with proliferation stages in the cell growth and regeneration models.
Conclusions :
We have identified the unique TAC entity and uncovered a group of cell cycle-dependent genes as TAC signature markers using single cell transcriptomics and HCEC culture models. The findings provide insight into SC/TAC concept and lay the foundation for understanding corneal homeostasis and diseases.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.