Single-Cell Transcriptomics Identifies Limbal Stem Cell Population and Cell Types Mapping Its Differentiation Trajectory in Limbal Basal Epithelium of Human Cornea

Authors

De-Quan Li
Sangbae Kim
Jin-Miao Li
Qianmiao Gao
Jongsu Choi
Fang Bian
Jiaoyue Hu
Yun Zhang
Jin Li
Rong Lu
Yumei Li
Stephen C Pflugfelder
Hongyu Miao
Rui Chen

Publication Date

4-1-2021

Journal

The Ocular Surface

DOI

10.1016/j.jtos.2020.12.004

PMID

33388438

PMCID

PMC8359589

PubMedCentral® Posted Date

4-1-2022

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Cell Differentiation, Cornea, Epithelium, Corneal, Humans, Limbus Corneae, Stem Cells, Transcriptome, Adult stem cells, Cornea, Epithelium, Limbal stem cells, Single-cell transcriptomics

Abstract

PURPOSE: This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution.

METHODS: Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models.

RESULTS: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness.

CONCLUSIONS: This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

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