Rabbit limbal epithelial side population (SP) cells and non-SP cells
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ABSTRACT:
ORGANISM(S): Oryctolagus cuniculus
PROVIDER: GSE79620 | GEO | 2016/04/18
SECONDARY ACCESSION(S): PRJNA316450
REPOSITORIES: GEO
Similar Datasets
Project description:Recently we had discovered a solid cord like structure at the limbus of human eyes, termed as the Limbal Epithelial Crypt (LEC). It arises from the undersurface of the interpalisade rete ridges and extends towards the conjunctiva over the conjunctival stroma. Anatomical and immunohistochemical studies have shown it to be potentially a Stem Cell Niche. To confirm this hypothesis we conducted comparative gene expression profile of LEC with pathway and Geneontology studies in comparison with other ocular surface epithelial regions such as cornea, limbus, LEC stroma and conjunctiva. Frozen tissue blocks of corneoscleral buttons dissected from cadaver eyes were cryosectioned. These tissue sections from different ocular surface regions were laser microdissected. Extracted RNA was amplified & hybridized to 30,000k Human spotted cDNA microarray chips. Raw data obtained with Genepix Pro6 software was filtered, normalized & analysed on BASE & Jexpresspro software. Unpaired T-Test, Significance Analysis of Microarrays were performed on the data. Database for Annotation, Visualisation, and Integrated Discovery (DAVID) (http://www.DAVID.niaid.nih.gov) and Ingenuity Pathway Analysis (IPA) was used to determine the enriched GO terms and pathways in the differentially expressed genes. Quantitative gene expression analysis (qPCR) and immunohistochemistry was performed on the genes of interest. Statistical analysis for real time PCR was performed on SPSS16 to determine the normalised expression of gene of interest on ocular surface regions. Samples were prepared from five human ocular surface epithelial regions such as Cornea, Limbus, LEC, LEC Stroma, Conjunctiva. There were four replicates in each groups except Cornea and Conjunctiva with 3 each. The Standard Probe (SP) was prepared from mixing equal amount of Corneal and conjunctival epithelial RNA followed by ethanol precipitation. Samples were labelled with Cy5 dye and Standard Probe with Cy3 Dye. These were mixed in equal amount to prepare hybrid probes which were then hybridised to microarray slide.
2010-03-23 | E-GEOD-19035 | biostudies-arrayexpress
Project description:The limbus is a unique anatomic structure that is highly innervated and vascularized yet sets the boundary for the avascularized cornea. The corneal epithelial stem cells are believed to reside at the limbus. The intrinsic and external molecular signals that modulate the differentiation and proliferation of the limbal stems cells are still largely unidentified because of a lack of known specific markers. In this study, we used microarray technology to identify the unique gene expression profile in the limbus by comparing directly to that of its immediate adjacent structures, the cornea and conjunctiva, in the vervet monkey (Chlorocebus aethiops sabaeus). Many new genes were found to be preferentially expressed in the limbus, and two new biological pathways, melanin metabolism and apoptosis, were among other previously known processes identified in the limbus. These findings may shed light on the molecular components of limbal stem cells and their niche. Experiment Overall Design: The study consisted of gene expression profile comparisons across three anatomical structures of the eye. The parts of the eye compared included the cornea, the conjunctiva, and the limbus.
2008-04-06 | E-GEOD-10683 | biostudies-arrayexpress
Project description:Limbal stem cells (LSCs), known as corneal epithelial stem cells, are located at the basal epithelial layer of the corneal limbus and serve an important function in maintaining the homeostasis of the corneal epithelium. Several putative molecular markers of LSCs have been previously identified. However, the specificity of these markers remains largely controversial. To address this gap in the current understanding of LSCs, we performed a transcriptome profiling of heterogeneous corneal limbal basal cells using single-cell transcriptomics technology to identify LSCs and their exclusive markers. We isolated limbal basal cells from two young donors, constructed scRNA-seq libraries, generated RNA-sequencing data using the 10x Genomics platform, and then finally obtained the transcriptome of about 18,000 individual single-cells using Cell ranger (https://10xgenomics.com). Next, we performed quality control, filtering and data integration using Seurat package (https://satijalab.org/seurat), about 16,400 cells were retained for further downstream analysis such as dimensional reduction, unsupervised clustering, and Differentially expressed gene selection, trajectory analysis and visualization. We identified 11 unique clusters of cells assigned to a putative cell type based on published known biomarkers for differentiation, proliferation and putative epithelial stem cells. As a results, we found 2 Terminally Differentiated Cell (TDC), 3 Post-Mitotic Cell (PMC), 1 Transient Amplifying Cell (TAC), 2 Limbal Progenitor Cell (LPC), Putative Limbal Stem Cell (LSC) and 2 Melanocyte (MC) sub-cell type clusters. Furthermore, we confirmed the trajectory order of LSC differentiation for 9 clusters using Pseudotemporal and Functional PCA analysis. Lastly, we validated each assigned cell subtypes and determined their location in human corneal limbus tissue with 9 markers using RNAscope We were able to reveal the heterogeneity of corneal limbal basal epithelium by defining novel dynamic trajectories for cell types in a pseudotemporal manner. This approach allowed us to identify the distinct clusters of LSCs and progenitors with exclusively expressed markers, and might apply for translational research on regenerating a normal corneal epithelium and restoring vision.
2021-01-14 | GSE153515 | GEO
Project description:To date, there is no specific marker for limbal epithelial stem cells. The identification of a marker that is expressed in the limbal epithelium but not in the cornea or conjunctiva epithelium has been a growing need. To search for limbal-specific marker(s), we performed preferential gene profiling in the limbus in direct comparison to that in the cornea and conjunctiva using microarray technique. This study consisted of gene expression profiles comparing the limbus to the cornea and conjunctiva.
2013-01-01 | E-GEOD-38190 | biostudies-arrayexpress
Project description:The limbus is a unique anatomic structure that is highly innervated and vascularized yet sets the boundary for the avascularized cornea. The corneal epithelial stem cells are believed to reside at the limbus. The intrinsic and external molecular signals that modulate the differentiation and proliferation of the limbal stems cells are still largely unidentified because of a lack of known specific markers. In this study, we used microarray technology to identify the unique gene expression profile in the limbus by comparing directly to that of its immediate adjacent structures, the cornea and conjunctiva, in the vervet monkey (Chlorocebus aethiops sabaeus). Many new genes were found to be preferentially expressed in the limbus, and two new biological pathways, melanin metabolism and apoptosis, were among other previously known processes identified in the limbus. These findings may shed light on the molecular components of limbal stem cells and their niche. Keywords: transcription profiling, gene expression
2008-03-05 | GSE10683 | GEO
Project description:Limbal stem cells including epithelial and stromal/Mesenchymal stem cells that contribute to sustained corneal homeostasis, maintain their ability to act as self-renewal progenitor cells by virtue of their limbal niche and intercellular communication. Extracellular vehicles (EVs), including exosomes (Exos), are important paracrine mediators through their cargo transfer for intercellular communication in various stem cell niches. Previously we have shown the differential cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos, in limbal epithelial cells (LEC) in normal (N) and diabetic (DM) limbal niche. In the present study, to have a comprehensive knowledge of reciprocal LEC-LSC crosstalk, we investigated the proteomics and miRNA profile of exosomes derived from LEC and their regulatory roles in LSC in N and DM limbus. Our study showed wound healing and proliferation rates in primary N-LSC were significantly enhanced upon treatment by normal LEC-derived Exos (N-Exos), but not by diabetic Exos (DM-Exos). Further, N-Exos treated LSC showed downregulation of keratocyte markers, ALDH3A1 and lumican, but not keratocan, and upregulation of MSC markers, CD105, CD90, and CD73 compared to the DM-Exos treated LSC. Using next generation sequencing (NGS) and proteomics analysis, we revealed some miRNAs and proteins in the Exos that affect the cellular crosstalk and the function of the cornea. We also documented differences in DM vs. normal LEC-derived Exo’s cargos. Overall, DM-Exos have less effect on LSC proliferation, wound healing, and stem cell maintenance than N-Exos, likely by transferring their cargo proteins and/or regulatory miRNAs targeting cell cycle, ERK/MAPK, TGF-β, EMT, PI3K-Akt-mTOR signaling molecules. This suggests that the small RNA and protein cargo differences in DM vs. N LEC-derived Exos could contribute to the disease state. Our study revealed a complex contribution of Exos to health and diabetic state of corneal homeostasis and suggests the potential of EV therapeutics for diabetic cornea regenerative medicine
2023-10-23 | PXD040918 | Pride
Project description:Limbal epithelial stem cells (LESCs) reside within the LSC niche (LSCN), located at the annular transition zone between the cornea and conjunctiva. LESCs are important for the long-term maintenance of the corneal epithelium and critical for repopulating the corneal epithelium after injury. We have recently identified that a hyaluronan (HA)-rich extracellular matrix (ECM) exists within the LSCN, and that this HA matrix is necessary for maintaining LESCs in the “stem cell” state. Herein we further characterized the composition of the LSCN, identifying key components of the HA-rich matrix. We identified that the hyaladherins IαI, TSG-6 and versican are highly expressed in the limbus when compared to the cornea. For IαI, Heavy chain 5 (HC5) was found to be the most highly expressed HC in the mouse and human cornea, and associates with HA forming HA/HC5 specific matrices. CD44 is the most likely receptor that mediates the interaction between LESCs and the HA-specific matrix. The LSCN is composed of a rich HA matrix that contains HA/HC5. HA/HC5 complexes could be used as an improved substrate for culturing LESCs during ex vivo expansion for limbal stem cell transplantation.
2023-11-08 | GSE244412 | GEO
Project description:To date, there is no specific marker for limbal epithelial stem cells. The identification of a marker that is expressed in the limbal epithelium but not in the cornea or conjunctiva epithelium has been a growing need. To search for limbal-specific marker(s), we performed preferential gene profiling in the limbus in direct comparison to that in the cornea and conjunctiva using microarray technique.
2013-01-01 | GSE38190 | GEO
Project description:Limbal epithelial stem cell (LESC) deficiency represents a significant clinical problem especially in bilateral cases. Induced pluripotent stem cells (iPSC) may be a promising source of LESC, allowing standardized and continual propagation and banking. The objective of this study was to generate iPSC from human limbal epithelial cultures and differentiate them back into limbal epithelial cells using substrata mimicking the natural LESC niche. Using Yamanaka’s episomal vectors limbal-derived iPSC were reprogrammed from LESC cultured from donor corneoscleral rims and from human skin fibroblasts. A clone from limbal-derived iPSC expressed stemness markers, had a diploid karyotype, and produced teratomas in nude mice representing three germ layers. Compared to parental LESC, this clone had fewer specific gene methylation changes revealed using the Illumina Infinium Methylation 450k Beadchips than compared to skin fibroblasts. The expression of putative LESC markers was examined by quantitative RT-PCR and immunostaining in limbal-derived and fibroblast-derived iPSC cultured on denuded human amniotic membrane or denuded cornea. Limbal-derived iPSC had markedly stronger expression of PAX6, ABCG2, Np63, keratins 14, 15, 17, and N-cadherin than fibroblast-derived iPSC. On denuded corneas, limbal-derived iPSC showed the expression of differentiated corneal keratins 3 and 12. The data suggest that iPSC differentiation to a desired lineage may be facilitated by their generation from the same tissue. This may be related to preservation of parental tissue epigenetic methylation signatures in iPSC and use of biological substrata similar to the natural niche of parental cells. The data pave the way for generating transplantable LESC from limbal-derived iPSC. Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip
2014-06-30 | E-GEOD-53918 | biostudies-arrayexpress
Project description:To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal and conjunctival cell types from development to adulthood. The conjunctival epithelium is the first to be specified in the epithelial layer , followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2,a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. Single cell RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal epithelial progenitor markers during limbal epithelial cell expansion: this phenomenon was independent of 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 TA cells in the corneal epithelium as two key mechanistic changes underlying the disease phenotype. Our scRNA-Seq data comparisons of developing and adult cornea and conjunctiva provide a unique resource for defining pathways/genes that could lead to improvement in ex vivo expansion methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders.
2021-04-19 | GSE155683 | GEO