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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 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 -
Experimental Eye Research Apr 2021The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be... (Review)
Review
The function of limbal stem/progenitor cells (LSCs) is critical to maintain corneal epithelial homeostasis. Many external insults and intrinsic defects can be deleterious to LSCs and their niche microenvironment, resulting in limbal stem cell dysfunction or deficiency (LSCD). Ocular comorbidities, frequent in eyes with LSCD, can exacerbate the dysfunction of residual LSCs, and limit the survival of transplanted LSCs. Clinical presentation and disease evolution vary among different etiologies of LSCD. New ocular imaging modalities and molecular markers are now available to standardize the diagnosis criteria and stage the severity of the disease. Medical therapies may be sufficient to reverse the disease if residual LSCs are present. A stepwise approach should be followed to optimize the ocular surface, eliminate the causative factors and treat comorbid conditions, before considering surgical interventions. Furthermore, surgical options are selected depending on the severity and laterality of the disease. The standardized diagnostic criteria to stage the disease is necessary to objectively evaluate and compare the efficacy of the emerging customized therapies.
Topics: Corneal Diseases; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cells
PubMed: 33571530
DOI: 10.1016/j.exer.2021.108437 -
Proceedings of the National Academy of... Jan 2023Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is...
Many epithelial compartments undergo constitutive renewal in homeostasis but activate unique regenerative responses following injury. The clear corneal epithelium is crucial for vision and is renewed from limbal stem cells (LSCs). Using single-cell RNA sequencing, we profiled the mouse corneal epithelium in homeostasis, aging, diabetes, and dry eye disease (DED), where tear deficiency predisposes the cornea to recurrent injury. In homeostasis, we capture the transcriptional states that accomplish continuous tissue turnover. We leverage our dataset to identify candidate genes and gene networks that characterize key stages across homeostatic renewal, including markers for LSCs. In aging and diabetes, there were only mild changes with <15 dysregulated genes. The constitutive cell types that accomplish homeostatic renewal were conserved in DED but were associated with activation of cell states that comprise "adaptive regeneration." We provide global markers that distinguish cell types in homeostatic renewal vs. adaptive regeneration and markers that specifically define DED-elicited proliferating and differentiating cell types. We validate that expression of SPARC, a marker of adaptive regeneration, is also induced in corneal epithelial wound healing and accelerates wound closure in a corneal epithelial cell scratch assay. Finally, we propose a classification system for LSC markers based on their expression fidelity in homeostasis and disease. This transcriptional dissection uncovers the dramatically altered transcriptional landscape of the corneal epithelium in DED, providing a framework and atlas for future study of these ocular surface stem cells in health and disease.
Topics: Mice; Animals; Limbus Corneae; Cell Differentiation; Cornea; Epithelium, Corneal; Wound Healing; Dry Eye Syndromes; Homeostasis
PubMed: 36595669
DOI: 10.1073/pnas.2204134120 -
Nature Communications Jan 2021Adult stem cell identity, plasticity, and homeostasis are precisely orchestrated by lineage-restricted epigenetic and transcriptional regulatory networks. Here, by...
Adult stem cell identity, plasticity, and homeostasis are precisely orchestrated by lineage-restricted epigenetic and transcriptional regulatory networks. Here, by integrating super-enhancer and chromatin accessibility landscapes, we delineate core transcription regulatory circuitries (CRCs) of limbal stem/progenitor cells (LSCs) and find that RUNX1 and SMAD3 are required for maintenance of corneal epithelial identity and homeostasis. RUNX1 or SMAD3 depletion inhibits PAX6 and induces LSCs to differentiate into epidermal-like epithelial cells. RUNX1, PAX6, and SMAD3 (RPS) interact with each other and synergistically establish a CRC to govern the lineage-specific cis-regulatory atlas. Moreover, RUNX1 shapes LSC chromatin architecture via modulating H3K27ac deposition. Disturbance of RPS cooperation results in cell identity switching and dysfunction of the corneal epithelium, which is strongly linked to various human corneal diseases. Our work highlights CRC TF cooperativity for establishment of stem cell identity and lineage commitment, and provides comprehensive regulatory principles for human stratified epithelial homeostasis and pathogenesis.
Topics: Adolescent; Adult; Adult Stem Cells; Aged; Cell Lineage; Cell Plasticity; Cells, Cultured; Child; Chromatin; Chromatin Immunoprecipitation Sequencing; Core Binding Factor Alpha 2 Subunit; Corneal Diseases; Epithelium, Corneal; Female; Gene Expression Regulation; Gene Knockdown Techniques; Gene Regulatory Networks; Humans; Limbus Corneae; Male; Middle Aged; PAX6 Transcription Factor; Primary Cell Culture; RNA-Seq; Smad3 Protein
PubMed: 33462242
DOI: 10.1038/s41467-020-20713-z -
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 -
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 -
Current Opinion in Ophthalmology Mar 2020Most microinvasive glaucoma surgery (MIGS) procedures bypass outflow resistance residing proximally in the trabecular meshwork and inner wall of Schlemm's canal. A novel... (Review)
Review
PURPOSE OF REVIEW
Most microinvasive glaucoma surgery (MIGS) procedures bypass outflow resistance residing proximally in the trabecular meshwork and inner wall of Schlemm's canal. A novel procedure combining trabeculotomy with viscodilation adds to this by also addressing distal resistance of the canal and collector channel ostia. This review examines the development and evidence for both trabeculotomy and canaloplasty separately and the combination in a single procedure.
RECENT FINDINGS
Recent aqueous angiography studies have confirmed the segmental nature of outflow through Schlemm's canal highlighting the need to address distal outflow pathway resistance. Combined trabeculotomy and viscodilation ab interno is a novel approach with a new purpose-designed device (OMNI Surgical System) becoming available to surgeons in early 2018. Recent results as both a standalone and combined with cataract procedure demonstrate significant intraocular pressure reductions with an average 41% reduction from baseline in the pseudophakic group.
SUMMARY
Targeting both distal as well as proximal points of outflow resistance in the conventional pathway may prove to be a highly efficacious MIGS modality. Additional large prospective studies are currently ongoing to confirm these preliminary results.
Topics: Aqueous Humor; Glaucoma, Open-Angle; History, 20th Century; History, 21st Century; Humans; Intraocular Pressure; Limbus Corneae; Trabecular Meshwork; Trabeculectomy
PubMed: 31904595
DOI: 10.1097/ICU.0000000000000639 -
Asia-Pacific Journal of Ophthalmology... Dec 2020Treatment of limbal stem cell deficiency is challenging. Multiple options can be adopted according to the underlying cause and the patient and physician preferences.... (Review)
Review
Treatment of limbal stem cell deficiency is challenging. Multiple options can be adopted according to the underlying cause and the patient and physician preferences. Stem cell transplant is a common treatment modality and several techniques have been described with outcomes varying by the laterality of the condition. Keratoprosthesis is a preferred option for bilateral conditions. Indications for type 1 and type 2 keratoprosthesis differ and the past 2 decades have seen a revolution in the field of keratoprosthesis with encouraging and improved outcomes. Management also includes preventive measures and measures to optimize/stabilize the ocular surface that would go a long way in reducing the deterioration of the ocular surface. The aim of this review is to provide an overview of the currently available techniques and to present a comprehensive algorithm to assist in decision making for unilateral and bilateral limbal stem cell deficiency.
Topics: Corneal Diseases; Humans; Limbus Corneae; Stem Cell Transplantation; Visual Acuity
PubMed: 33323706
DOI: 10.1097/APO.0000000000000326 -
Progress in Retinal and Eye Research Nov 2021The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus.... (Review)
Review
The corneal epithelium is continuously renewed by limbal stem/progenitor cells (LSCs), a cell population harbored in a highly regulated niche located at the limbus. Dysfunction and/or loss of LSCs and their niche cause limbal stem cell deficiency (LSCD), a disease that is marked by invasion of conjunctival epithelium into the cornea and results in failure of epithelial wound healing. Corneal opacity, pain, loss of vision, and blindness are the consequences of LSCD. Successful treatment of LSCD depends on accurate diagnosis and staging of the disease and requires restoration of functional LSCs and their niche. This review highlights the major advances in the identification of potential LSC biomarkers and components of the LSC niche, understanding of LSC regulation, methods and regulatory standards in bioengineering of LSCs, and diagnosis and staging of LSCD. Overall, this review presents key points for researchers and clinicians alike to consider in deepening the understanding of LSC biology and improving LSCD therapies.
Topics: Bioengineering; Corneal Diseases; Epithelium, Corneal; Humans; Limbus Corneae; Stem Cells
PubMed: 33676006
DOI: 10.1016/j.preteyeres.2021.100956