Single-Cell Transcriptomics Identifies a Unique Entity and Signature Markers of Transit-Amplifying Cells in Human Corneal Limbus

Abstract

PURPOSE. Differentiated from adult stem cells (ASCs), transit-amplifying cells (TACs) play an important role in tissue homeostasis, development, and regeneration. This study aimed to characterize the gene expression profile of a candidate TAC population in limbal basal epithelial cells using single-cell RNA sequencing (scRNA-seq). METHODS.Single cells isolated from the basal corneal limbus were subjected to scRNA-seq using the 10x Genomics platform. Cell types were clustered by graph-based visualization methods and unbiased computational analysis. BrdU proliferation assays, immunofluorescent staining, and real-time reverse transcription quantitative polymerase chain reaction were performed using multiple culture models of primary human limbal epithelial cells to characterize the TAC pool. 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 a 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 revealed to be largely enriched by the TAC population. The 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 in a time-and cycle-dependent manner with proliferation stages in the cell growth and regeneration models. CONCLUSIONS.For the first time, to the best of our knowledge, we have identified a unique TAC entity and uncovered a group of cell cycle-dependent genes that serve as TAC signature markers. The findings provide insight into ASCs and TACs and lay the foundation for understanding corneal homeostasis and diseases.

Single cell transcriptomics reveals the heterogeneity of the human cornea to identify novel markers of the limbus and stroma

“…Interestingly, primary limbal cells expressing a lower level amount of ΔNp63 and p63α, based on fluorescence intensity, also showed a reduced expression of caveolin-1 and cyclin-dependent kinase 2 respectively (Supplementary Figures S4 and S6 ). Interestingly, two recent studies also reported STMN1 and UBE2C to be specific markers of limbal transit amplifying cells 43 , 45 . Finally, in a 2019 single cell transcriptome study, Kaplan et al hypothesized that the stemness of mouse limbal epithelial stem cells could be regulated through autophagy 46 .…”

Commentary: Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity In Vitro and In Vivo

“…Single-cell RNA-seq (scRNA-seq) technologies are broadly applied to dissect the cellular heterogeneity and expression dynamics, providing unprecedented insights into single-cell biology (Plass et al, 2018;Li et al, 2020). A few groups have recently used scRNA-seq to analyze LSC populations and corneal homeostasis (Li et al, 2021a;Li et al, 2021b). These data provide a new avenue for studying the mechanisms of LSC self-renewal (Altshuler et al, 2021).…”

Limbal BCAM expression identifies a proliferative progenitor population capable of holoclone formation and corneal differentiation

“…Limbal TACs are considered to be ''early'' or immature due to their relatively undifferentiated phenotype, as opposed to ''late'' or mature central cornea TACs, which begin expressing corneal differentiation markers such as KRT12 (Kaplan et al, 2019;Lehrer et al, 1998). Recently, single-cell RNA sequencing (scRNA-seq) analyses of mixed corneal populations were performed and have begun to shed more light on corneal epithelial heterogeneity, including the LSC niche (Catala et al, 2021;Collin et al, 2021;Kaplan et al, 2019;Li et al, 2021).…”