June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Single-Cell Transcriptomics Identifies Limbal Stem Cell Population, Differentiation Trajectory and Signature Markers in Human Corneal Limbus
Author Affiliations & Notes
  • De-Quan Li
    Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Jinmiao Li
    Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China
  • Sangbae Kim
    HGSC, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States
  • Jongsu Choi
    HGSC, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States
  • Fang Bian
    Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Stephen C Pflugfelder
    Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, Texas, United States
  • Rui Chen
    HGSC, Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States
  • Footnotes
    Commercial Relationships   De-Quan Li, None; Jinmiao Li, None; Sangbae Kim, None; Jongsu Choi, None; Fang Bian, None; Stephen Pflugfelder, None; Rui Chen, None
  • Footnotes
    Support  NIH NEI Grants EY023598 (DQL) and EY011915 (SCP), Core Grant for Vision Research EY002520, Lions Foundation for Sight, Research to Prevent Blindness, Oshman Foundation, William Stamps Farish Fund.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 861. doi:
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      De-Quan Li, Jinmiao Li, Sangbae Kim, Jongsu Choi, Fang Bian, Stephen C Pflugfelder, Rui Chen; Single-Cell Transcriptomics Identifies Limbal Stem Cell Population, Differentiation Trajectory and Signature Markers in Human Corneal Limbus. Invest. Ophthalmol. Vis. Sci. 2021;62(8):861.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Human corneal epithelial stem cells or limbal stem cells (LSCs), have been recognized to locate in corneal limbus for three decades. However, the molecular identity and definitive markers of LSCs are still elusive. This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC population 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 Uniform Manifold Approximation and Projection (UMAP) and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using ex vivo donor corneal tissues and in vitro culture models of primary human limbal epithelial cells (HLECs).

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 top expressed genes known as markers of 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 immunofluorescent staining, as well as their 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 single-cell transcriptomics 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.

This is a 2021 ARVO Annual Meeting abstract.

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