Project description:Here, we evaluate the efficacy of cryopreserved human embryonic stem cell (hESC)-derived corneal endothelial cells (CECs) to form a functional monolayer of corneal endothelium (CE) in mammals (rabbits) and non-human primates (monkeys). We injected cryopreserved hESC-derived CECs in rabbits and monkeys either immediately after removing 8 mm of the central portion of the CE or a few days later when corneal edema developed. All clinical models developed deturgesced and clear corneas 2-3 weeks following the CEC injection and remained comparable to the CE of the untreated eye. Confocal scanning microscopy confirmed an intact structure of hexagonal/polygonal cells and immunohistochemical analysis illustrated a monolayer expressing barrier and pump function proteins in the regenerated CE. The necropsy examination confirmed no remarkable change in multiple tissues examined for teratoma formation. In conclusion, our data demonstrate the efficacy of cryopreserved hESC-derived CECs to form a functional CE on the denuded Descemet’s membrane.
Project description:Corneal organoids are useful tools for disease modeling and tissue transplantation however they have not yet been well studied during maturation. We characterized human iPSC derived corneal organoids at 1, 2, 3 and 4 months of development using single-cell RNA sequencing to determine the cellular heterogeneity at each stage. We found pluripotent cell clusters committed to epithelial cell lineage at 1 month early corneal epithelial, endothelial and stromal cells markers at 2 months, keratocytes as the largest cell population at 3 months, and a large epithelial cell population at 4 months. We compared organoid to fetal corneal development at different stages and found that 4 month organoids closely resemble the corneal cellular complexity of the fetal (16 post conception week) and adult cornea. Using RNA velocity pseudotime trajectory analysis, we found that eye field primordial cells and limbal stem cells appear to give rise to corneal epithelial cells during development.
Project description:In this study, we utilized single-cell RNA sequencing (scRNA-seq) to investigate the effects of tacrolimus and rapamycin on corneal graft survival following penetrating keratoplasty in mice. Post-transplantation, the recipient mice were administered daily intraperitoneal injections of tacrolimus, rapamycin, or PBS. Fifteen days after treatment, scRNA-seq was employed to detect molecular and cellular changes within the corneal tissues.
Project description:Col8a2 mutation is one of the genetic causes of Fuchs endothelial corneal dystrophy (FECD). The Col8a2Q455K homozygous mutant mouse serves as a disease model for FECD. Corneal endothelial cells were isolated from early-stage (≤ 2 months old) and late-stage (≥ 8 months old) Col8a2Q455K homozygous mutant mice and their age-matched wild-type controls. This study investigates transcriptomic alterations associated with FECD progression caused by the Col8a2 mutation.
Project description:Transplantation of amniotic membrane-expanded limbal epithelium (AMLE) in place of donor tissue grafts results in significantly improved outcomes for patients suffering from severe limbal stem cell deficiency; however the reasons for such superior results are unclear. The purpose of this study was to identify transcriptional gene profiles specific to AMLE and donor central corneal epithelium (CE), which may contribute to the divergent clinical outcomes observed following transplant. Limbal fibroblasts which underlie the epithelium and secrete extracellular matrix proteins following injury/surgery were also profiled. Using cell culture, immunofluorescence, microarray gene expression profiling and qRT-PCR validation; this study aims to identify enriched biological processes and pathways which characterise AMLE and CE tissues. We hope the study outcomes will shed light onto the factors which contribute to provide the improved clinical outcomes associated with AMLE transplantation. Gene expression profiling of three central corneal buttons, three amniotic membrane-expanded limbal epithelial cultures and three limbal fibroblast cultures
Project description:Keratoconus (KC) is a corneal ectasia characterized by structural changes, resulting in progressive thinning and biomechanical weakening that can lead to worsening visual acuity due to irregular astigmatism. Corneal collagen Crosslinking (CXL) and Intracorneal Ring Segment (ICRS) are widely used treatments in KC disease, but the alterations they cause in biomechanical mediators are still poorly understood. The aim of this study was to analyze the tear proteome profile before and after treatments to identify biomarkers altered by surgery.
Project description:MicroRNA profile comparison of the corneal endothelium of young and old mice: implications for senescence of the corneal endothelium We collected the corneal endothelia from 30 mice aged 10-13 weeks and the corneal endothelia from 30 mice aged 2 years. The samples were pooled into six groups (y1, y2, y3 and s1, s2, s3). Each group comprised corneal endothelia from ten mice, and these six groups were used for a genome-wide microRNA microarray study.
Project description:The cornea is the most innervated tissue in the human body. Myelinated axons upon inserting into the peripheral corneal stroma lose their myelin sheaths and continue into the central cornea wrapped by only nonmyelinating corneal SCs (nm-cSCs). This anatomical organization is believed to be important for central vision. Here we employed single-cell RNA sequencing (scRNA-seq), microscopy, and transgenics to characterize these nm-cSCs of the central cornea. Using principal component analysis, uniform manifold approximation and projection, and unsupervised hierarchal cell clustering of scRNA-seq data derived from central corneal cells of male rabbits, we successfully identified several clusters representing different corneal cell types, including a unique cell cluster representing nm-cSCs. To confirm protein expression of cSC genes, we performed cross-species validation, employing corneal whole mount immunostaining with confocal microscopy in mouse corneas. We expect that our results will advance the future study of nm-cSCs in applications of nerve repair, and provide a resource for the study of corneal sensory function.
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.
Project description:The specific genes that distinguish normal fracture healing from abnormal healing or nonunion in humans are unknown. This study was an exploratory investigation of peripheral blood from 2 chronic nonunion patients collected perioperatively (pre/post revision surgery) and at 3 months post revision follow up for comparison to Acutely injured subjects and Healthy volunteer cohorts analyzed separately. We used microarrays to do a global comparison between 2 chronic nonunion patients collected perioperatively (pre/post revision surgery) and at 3 months post revision follow up.