Project description:Whole human conjunctivae and human corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and processed between 48 - 72 hr of death. No donor details apart from age, sex and cause of death were released. Criteria for inclusion were donor ages between 25 - 65 years old and overt physical integrity of the epithelium over the central cornea as revealed by a quick stain with 0.1 % Trypan blue. For each replicate experiment (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3), RNA from two different corneal or conjunctival specimens was pooled and converted into cDNA. Appropriately fragmented biotin-labeled cRNA was hybridized to the HG-U133A GeneChips® (Affymetrix) oligonucleotide microarray following the manufacture protocol. Three separate experiments each including one conjunctival and one corneal cRNA pool were performed (Conjunctiva 1 and Cornea 1; Conjunctiva 2 and Cornea 2;Conjunctiva 3 and Cornea 3).
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.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
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.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
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.
Project description:The identification of a marker that is expressed in the conjunctival epithelium but not in the corneal epithelium has been a growing need. A more specific marker of limbal and conjunctival epithelia would be necessary to detect non-corneal epithelial cells on the corneal surface. To search for conjunctival specific marker(s), we first performed preferential gene profiling in the conjunctiva in direct comparison to that in the cornea using microarray technique. The study consisted of gene expression profiles comparing the cornea and conjunctiva.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs. One-condition experment, gene expression of 3A6