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

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      The Ocular Surface
      Elsevier Inc
      Embryonic and fetal eye, Cornea, Conjunctiva, Ocular surface, Single cell RNA-Seq, Single cell ATAC-Seq, Limbal stem cells (LSCs), Limbal progenitor cells (LPCs), Limbal epithelial cells (LECs), LSCs dysplasia, Keratoconus, Limbal epithelial expansion

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          Abstract

          Purpose

          Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.

          Methods

          Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs).

          Results

          scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC 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 limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated 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 LPCs, which predate the formation of limbal niche by a few weeks.

          Conclusions

          Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

          Graphical abstract

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

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          Most cited references92

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          Comprehensive Integration of Single-Cell Data

          Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.
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            CellPhoneDB: inferring cell–cell communication from combined expression of multi-subunit ligand–receptor complexes

            Cell-cell communication mediated by ligand-receptor complexes is critical to coordinating diverse biological processes, such as development, differentiation and inflammation. To investigate how the context-dependent crosstalk of different cell types enables physiological processes to proceed, we developed CellPhoneDB, a novel repository of ligands, receptors and their interactions. In contrast to other repositories, our database takes into account the subunit architecture of both ligands and receptors, representing heteromeric complexes accurately. We integrated our resource with a statistical framework that predicts enriched cellular interactions between two cell types from single-cell transcriptomics data. Here, we outline the structure and content of our repository, provide procedures for inferring cell-cell communication networks from single-cell RNA sequencing data and present a practical step-by-step guide to help implement the protocol. CellPhoneDB v.2.0 is an updated version of our resource that incorporates additional functionalities to enable users to introduce new interacting molecules and reduces the time and resources needed to interrogate large datasets. CellPhoneDB v.2.0 is publicly available, both as code and as a user-friendly web interface; it can be used by both experts and researchers with little experience in computational genomics. In our protocol, we demonstrate how to evaluate meaningful biological interactions with CellPhoneDB v.2.0 using published datasets. This protocol typically takes ~2 h to complete, from installation to statistical analysis and visualization, for a dataset of ~10 GB, 10,000 cells and 19 cell types, and using five threads.
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              Global estimates of visual impairment: 2010.

              From the most recent data the magnitude of visual impairment and its causes in 2010 have been estimated, globally and by WHO region. The definitions of visual impairment are the current definitions of presenting vision in the International Classification of Diseases version 10. A systematic review was conducted of published and unpublished surveys from 2000 to the present. For countries without data on visual impairment, estimates were based on newly developed imputation methods that took into account country economic status as proxy. Surveys from 39 countries satisfied the inclusion criteria for this study. Globally, the number of people of all ages visually impaired is estimated to be 285 million, of whom 39 million are blind, with uncertainties of 10-20%. People 50 years and older represent 65% and 82% of visually impaired and blind, respectively. The major causes of visual impairment are uncorrected refractive errors (43%) followed by cataract (33%); the first cause of blindness is cataract (51%). This study indicates that visual impairment in 2010 is a major health issue that is unequally distributed among the WHO regions; the preventable causes are as high as 80% of the total global burden.
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                Author and article information

                Contributors
                Journal
                Ocul Surf
                Ocul Surf
                The Ocular Surface
                Elsevier Inc
                1542-0124
                1937-5913
                1 July 2021
                July 2021
                : 21
                : 279-298
                Affiliations
                [a ]Biosciences Institute, Faculty of Medical Sciences, Newcastle University, UK
                [b ]Newcastle Cellular Therapies Facility, Newcastle University and Newcastle Upon Tyne Hospitals NHS Foundation Trust, UK
                [c ]Department of Genetics and Developmental Biology, The Ruth and Bruce Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Israel
                [d ]NHS Blood and Transplant Tissue and Eye Services, Liverpool, UK
                [e ]Sunderland Eye Infirmary, South Tyneside and Sunderland NHS Foundation Trust, Sunderland, UK
                [f ]UK Department of Ophthalmology, Royal Victoria Infirmary and Newcastle University, Newcastle, UK
                Author notes
                []Corresponding author. majlinda.lako@ 123456ncl.ac.uk
                [∗∗ ]Corresponding author. lyle.armstrong@ 123456ncl.ac.uk
                [1]

                Present address: Microscopy Centre and Department of Applied Clinical Sciences and Biotechnology, University of L'Aquila, Italy.

                [2]

                joint first authors.

                [3]

                joint second authors.

                Article
                S1542-0124(21)00021-5
                10.1016/j.jtos.2021.03.010
                8343164
                33865984
                3e55e1f8-ca22-4c48-91e4-eec607758954
                © 2021 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 15 December 2020
                : 5 March 2021
                : 25 March 2021
                Categories
                High Impact Original Research

                embryonic and fetal eye,cornea,conjunctiva,ocular surface,single cell rna-seq,single cell atac-seq,limbal stem cells (lscs),limbal progenitor cells (lpcs),limbal epithelial cells (lecs),lscs dysplasia,keratoconus,limbal epithelial expansion

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