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A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

Joseph Collin, Rachel Queen, Darin Zerti, Sanja Bojic, Nicky Moyse, Marina Moya Molina, Chunbo Yang, Gary Reynolds, Rafiqul Hussain, Jonathan M Coxhead, Steven Lisgo, Deborah Henderson, Agatha Joseph, Paul Rooney, Saurabh Ghosh, Che Connon, Muzlifah Haniffa, Francisco Figueiredo, Lyle Armstrong, Majlinda Lako
doi: https://doi.org/10.1101/2020.07.09.195438
Joseph Collin
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Rachel Queen
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Darin Zerti
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
2Microscopy Centre and Department of Applied Clinical Sciences and Biotechnology, University of L’Aquila, Italy
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Sanja Bojic
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Nicky Moyse
3Newcastle Cellular Therapies Facility, Newcastle University and Newcastle upon Tyne Hospitals NHS Foundation Trust, UK
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Marina Moya Molina
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Chunbo Yang
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Gary Reynolds
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Rafiqul Hussain
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Jonathan M Coxhead
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Steven Lisgo
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Deborah Henderson
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Agatha Joseph
4NHS Blood and Transplant Tissue and Eye Services, Liverpool, UK
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Paul Rooney
4NHS Blood and Transplant Tissue and Eye Services, Liverpool, UK
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Saurabh Ghosh
5Royal Victoria Infirmary, Sunderland, UK
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Che Connon
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Muzlifah Haniffa
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Francisco Figueiredo
6Department of Ophthalmology, Royal Victoria Infirmary and Newcastle University, Newcastle, UK
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Lyle Armstrong
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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Majlinda Lako
1Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
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  • For correspondence: majlinda.lako@ncl.ac.uk
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Summary

To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA- and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders.

Key findings

  • scRNA-Seq of adult human cornea and conjunctiva reveals the signature of various ocular surface cell populations

  • scRNA-Seq of human developing cornea identifies stage-specific definitions of corneal epithelial, stromal and endothelial layers

  • scRNA-Seq analysis results in identification of novel markers for qLSCs and TA cells

  • Combined scRNA- and ATAC-Seq analysis reveals key transcriptional networks in qLSCs and TA cells and close interactions with immune cells

  • Expansion of limbal epithelium results in downregulation of qLSCs and acquisition of proliferative limbal epithelial progenitor markers

  • scRNA-Seq of keratoconus corneas reveals activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium

Figure1

Schematic presentation of main techniques and findings presented in this manuscript.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 10, 2020.
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A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche
Joseph Collin, Rachel Queen, Darin Zerti, Sanja Bojic, Nicky Moyse, Marina Moya Molina, Chunbo Yang, Gary Reynolds, Rafiqul Hussain, Jonathan M Coxhead, Steven Lisgo, Deborah Henderson, Agatha Joseph, Paul Rooney, Saurabh Ghosh, Che Connon, Muzlifah Haniffa, Francisco Figueiredo, Lyle Armstrong, Majlinda Lako
bioRxiv 2020.07.09.195438; doi: https://doi.org/10.1101/2020.07.09.195438
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A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche
Joseph Collin, Rachel Queen, Darin Zerti, Sanja Bojic, Nicky Moyse, Marina Moya Molina, Chunbo Yang, Gary Reynolds, Rafiqul Hussain, Jonathan M Coxhead, Steven Lisgo, Deborah Henderson, Agatha Joseph, Paul Rooney, Saurabh Ghosh, Che Connon, Muzlifah Haniffa, Francisco Figueiredo, Lyle Armstrong, Majlinda Lako
bioRxiv 2020.07.09.195438; doi: https://doi.org/10.1101/2020.07.09.195438

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