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The Ocular Surface Jul 2021Single 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...
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.
Topics: Adult; Cell Differentiation; Cells, Cultured; Cornea; Epithelial Cells; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cells
PubMed: 33865984
DOI: 10.1016/j.jtos.2021.03.010 -
Cell Stem Cell Jul 2021The accessibility and transparency of the cornea permit robust stem cell labeling and in vivo cell fate mapping. Limbal epithelial stem cells (LSCs) that renew the...
The accessibility and transparency of the cornea permit robust stem cell labeling and in vivo cell fate mapping. Limbal epithelial stem cells (LSCs) that renew the cornea are traditionally viewed as rare, slow-cycling cells that follow deterministic rules dictating their self-renewal or differentiation. Here, we combined single-cell RNA sequencing and advanced quantitative lineage tracing for in-depth analysis of the murine limbal epithelium. These analysis revealed the co-existence of two LSC populations localized in separate and well-defined sub-compartments, termed the "outer" and "inner" limbus. The primitive population of quiescent outer LSCs participates in wound healing and boundary formation, and these cells are regulated by T cells, which serve as a niche. In contrast, the inner peri-corneal limbus hosts active LSCs that maintain corneal epithelial homeostasis. Quantitative analyses suggest that LSC populations are abundant, following stochastic rules and neutral drift dynamics. Together these results demonstrate that discrete LSC populations mediate corneal homeostasis and regeneration.
Topics: Animals; Cornea; Homeostasis; Limbus Corneae; Mice; Stem Cells; Wound Healing
PubMed: 33984282
DOI: 10.1016/j.stem.2021.04.003 -
The Ocular Surface Apr 2021This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution.
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.
Topics: Cell Differentiation; Cornea; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cells; Transcriptome
PubMed: 33388438
DOI: 10.1016/j.jtos.2020.12.004 -
Seminars in Immunology May 2023Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the... (Review)
Review
Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production.
Topics: Humans; Fungi; Cornea; Keratitis; Fusarium; Neutrophils
PubMed: 37060806
DOI: 10.1016/j.smim.2023.101753 -
Stem Cell Reports Dec 2023Recently, the murine cornea has reemerged as a robust stem cell (SC) model, allowing individual SC tracing in living animals. The cornea has pioneered seminal... (Review)
Review
Recently, the murine cornea has reemerged as a robust stem cell (SC) model, allowing individual SC tracing in living animals. The cornea has pioneered seminal discoveries in SC biology and regenerative medicine, from the first corneal transplantation in 1905 to the identification of limbal SCs and their transplantation to successfully restore vision in the early 1990s. Recent experiments have exposed unexpected properties attributed to SCs and progenitors and revealed flexibility in the differentiation program and a key role for the SC niche. Here, we discuss the limbal SC model and its broader relevance to other tissues, disease, and therapy.
Topics: Mice; Animals; Epithelium, Corneal; Limbus Corneae; Cornea; Stem Cells; Cell Differentiation; Stem Cell Transplantation
PubMed: 38039972
DOI: 10.1016/j.stemcr.2023.10.023 -
Asia-Pacific Journal of Ophthalmology... Dec 2020Bowman layer (BL) transplantation has been described for various indications, including the treatment of postrefractive corneal haze, postherpetic scarring, and... (Review)
Review
Bowman layer (BL) transplantation has been described for various indications, including the treatment of postrefractive corneal haze, postherpetic scarring, and keratoconus. The unique properties of the BL explain its versatility. Robust, thin, and acellular, transplanted BL inhibits stromal scarring, flattens ectatic corneas, and poses minimal risk of immune reaction. This article reviews the history of BL transplantation and recent developments, and anticipated next steps for the procedure.
Topics: Cornea; Corneal Topography; Corneal Transplantation; Humans; Keratoconus; Visual Acuity
PubMed: 33156015
DOI: 10.1097/APO.0000000000000336 -
Journal of Internal Medicine Sep 2016The cornea is the transparent front part of the eye that transmits light to the back of the eye to generate vision. Loss of corneal transparency, if irreversible, leads... (Review)
Review
The cornea is the transparent front part of the eye that transmits light to the back of the eye to generate vision. Loss of corneal transparency, if irreversible, leads to severe vision loss or blindness. For decades, corneal transplantation using human donor corneas has been the only option for treating corneal blindness. Despite recent improvement in surgical techniques, donor cornea transplantation remains plagued by risks of suboptimal optical results and visual acuity, immune rejection and eventually graft failure. Furthermore, the demand for suitable donor corneas is increasing faster than the number of donors, leaving thousands of curable patients untreated worldwide. Here, we critically review the state of the art of biomaterials for corneal regeneration. However, the lessons learned from the use of the cornea as a disease model will allow for extension of the biomaterials and techniques for regeneration of more complex organs such as the heart.
Topics: Biomimetic Materials; Cornea; Corneal Transplantation; Humans; Prostheses and Implants; Prosthesis Design; Regeneration; Tissue Engineering
PubMed: 27098482
DOI: 10.1111/joim.12502 -
Investigative Ophthalmology & Visual... Oct 2023To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas.
PURPOSE
To generate a single-cell RNA-sequencing (scRNA-seq) map and construct cell-cell communication networks of mouse corneas.
METHODS
C57BL/6 mouse corneas were dissociated to single cells and subjected to scRNA-seq. Cell populations were clustered and annotated for bioinformatic analysis using the R package "Seurat." Differential expression patterns were validated and spatially mapped with whole-mount immunofluorescence staining. Global intercellular signaling networks were constructed using CellChat.
RESULTS
Unbiased clustering of scRNA-seq transcriptomes of 14,732 cells from 40 corneas revealed 17 cell clusters of six major cell types: nine epithelial cell, three keratocyte, two corneal endothelial cell, and one each of immune cell, vascular endothelial cell, and fibroblast clusters. The nine epithelial cell subtypes included quiescent limbal stem cells, transit-amplifying cells, and differentiated cells from corneas and two minor conjunctival epithelial clusters. CellChat analysis provided an atlas of the complex intercellular signaling communications among all cell types.
CONCLUSIONS
We constructed a complete single-cell transcriptomic map and the complex signaling cross-talk among all cell types of the cornea, which can be used as a foundation atlas for further research on the cornea. This study also deepens the understanding of the cellular heterogeneity and heterotypic cell-cell interaction within corneas.
Topics: Mice; Animals; Transcriptome; Mice, Inbred C57BL; Cornea; Epithelial Cells; Cell Communication
PubMed: 37792336
DOI: 10.1167/iovs.64.13.5 -
Nature Communications Apr 2020In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path...
In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path full-field/spectral-domain OCT microscope (FF/SD OCT), which enables cell-detail imaging of the entire ocular surface in humans (central and peripheral cornea, limbus, sclera, tear film) without contact and in real-time. Real-time performance is achieved through rapid axial eye tracking and simultaneous defocusing correction. Images contain cells and nerves, which can be quantified over a millimetric field-of-view, beyond the capability of IVCM and conventional OCT. In the limbus, palisades of Vogt, vessels, and blood flow can be resolved with high contrast without contrast agent injection. The fast imaging speed of 275 frames/s (0.6 billion pixels/s) allows direct monitoring of blood flow dynamics, enabling creation of high-resolution velocity maps. Tear flow velocity and evaporation time can be measured without fluorescein administration.
Topics: Adult; Angiography; Biomedical Engineering; Blood Flow Velocity; Cornea; Equipment Design; Female; Humans; Limbus Corneae; Male; Microscopy; Optical Imaging; Software; Tomography, Optical Coherence; Young Adult
PubMed: 32313067
DOI: 10.1038/s41467-020-15792-x -
Indian Journal of Ophthalmology Dec 2020Over the last decade, refractive surgery has been revolutionized by advancements in ablation profiles, available for the treatment of both regular and irregular corneas.... (Review)
Review
Over the last decade, refractive surgery has been revolutionized by advancements in ablation profiles, available for the treatment of both regular and irregular corneas. Advances in corneal imaging have helped highlight the presence of higher-order aberrations, the correction of which could result in a better quality of vision. Topographic measurements being static are more repeatable and pupil independent and therefore provide the ideal platform for correction of both lower and higher-order aberrations and could result in improved visual quality even in patients with seemingly regular corneas. The combination of topography-guided treatment with collagen cross-linking has further increased the scope of treating irregular corneas like keratoconus, post-laser in-situ keratomileusis ectasia, and pellucid marginal degeneration. This review delves into the current literature and guidelines available for the topographic treatment of regular and irregular corneas.
Topics: Collagen; Cornea; Corneal Topography; Humans; Keratoconus; Keratomileusis, Laser In Situ; Lasers, Excimer
PubMed: 33229645
DOI: 10.4103/ijo.IJO_2119_20