Single-cell multi-omics analysis of the early wound-responsive programs in mouse corneal epithelium
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1203210 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31834000.fastq.gz | Fastqsanger.gz | |||
| SRR31834001.fastq.gz | Fastqsanger.gz | |||
| SRR31834002.fastq.gz | Fastqsanger.gz | |||
| SRR31834003.fastq.gz | Fastqsanger.gz | |||
| SRR31834004.fastq.gz | Fastqsanger.gz |
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Project description:Wound healing is crucial for restoring homeostasis in living organisms. Although wound response mechanisms have been studied extensively, the gene regulatory programs involved remain to be elucidated. Here, we used single-cell RNA-seq and ATAC-seq analysis to profile the regulatory landscape of mouse corneal epithelium in early wound response.
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Project description:The cornea is the transparent tissue on the anterior surface of the eye. It is notably composed of a squamous stratified epithelium that forms a barrier protecting the ocular chamber. As the cornea is avascular, the delivery of nutrients and growth factors, required for corneal physiology, is essential. These nutrients and factors come from 1) the epithelium itself, 2) the innervation, and 3) the tear film, which together form the corneal microenvironment. Disturbances of this microenvironment, in the context of injury, aging or corneal pathology, can lead to progressive corneal opacification, resulting in blindness. More than 28 million people suffer from mono- or bilateral corneal blindness, making it the fourth cause of blindness worldwide. Corneal abrasion is the most common eye injury encountered in clinics, but poorly studied, making it a major issue in ophthalmology. It is characterized by a transient rupture in the integrity and cellular cohesion of the epithelial barrier, which are brought back thanks to a corneal wound healing process. The aim of this study is therefore to highlight the molecular impact of corneal abrasion on the microenvironment, and more specifically on the tear film. We performed a longitudinal study to identify a specific proteome of tears before and after corneal abrasion in mice. The corneal abrasion was performed unilaterally on female mice. Tear fluid samples were collected from the wounded eye and the contralateral eye before corneal abrasion and during the wound healing process up to 24hrs post-abrasion.
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Project description:Corneal injuries remain a major cause of consultation in the ophthalmology clinics worldwide. Repair of corneal wounds is a complex mechanism that involves cell death, migration, proliferation, differentiation, and extracellular matrix (ECM) remodeling. In the present study, we used a tissue-engineered, two-layers (epithelium and stroma) human cornea as a biomaterial to study both the cellular and molecular mechanisms of wound healing. Gene profiling on microarrays revealed important alterations in the pattern of genes expressed by tissue-engineered corneas in response to wound healing. Expression of many MMPs-encoding genes was shown by microarray and qPCR analyses to increase in the migrating epithelium of wounded corneas. Many of these enzymes were converted into their enzymatically active form as wound closure proceeded. In addition, expression of MMPs by human corneal epithelial cells (HCECs) was affected both by the stromal fibroblasts and the collagen-enriched ECM they produce. Most of all, results from mass spectrometry analyses provided evidence that a fully stratified epithelium is required for proper synthesis and organization of the ECM on which the epithelial cells adhere. In conclusion, and because of the many characteristics it shares with the native cornea, this human two layers corneal substitute may prove particularly useful to decipher the mechanistic details of corneal wound healing. Primary cultures of human corneal epithelial cells cultivated on BSA (number of replicates: 7), Collagen type I (number of replicates: 2), Collagen type IV (number of replicates: 2), Fibronectin (number of replicates: 2), Tenascin C (number of replicates: 2) and Laminin (number of replicates: 2) matrix. Central, internal and external ring of wounded Tissue-engineered human cornea.
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Project description:The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of metabolism and determine the transportation of endogenous and exogenous molecules across the corneal epithelium barrier. This study introduces multiple corneal epitheliums on a chip namely, Corneal Epithelium on a Chip (CEpOC), which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. We observed the spatiotemporal secretion of biologically relevant metabolites (i.e., antioxidant, glutathione and uric acid) as well as the depletion of essential nutrients such as amino acids and vitamins mimicking the in vivo molecules trafficking across the human corneal epithelium. Through the shifts of extracellular metabolites and quantitative analysis of mRNA associated with transporters, we were able to investigate the secretion and transportation activities across the polarized barrier in a correlation with the expression of corneal transporters. Thus, CEpOC can provide a non-invasive, simple, yet effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as advanced experimental model of the human corneal epithelium.
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