ABSTRACT: Neutrophil infiltration occurres in two phases during sterile inflammation to cornea: small initial phase (Phase I) that began within 15 min, and larger second phase (Phase II) that peaked at 24-48 h. Microarrays were used to identify candidate stimuli that initiated the two phases.
Project description:Considerable interest has been generated for the development through cell-tissue engineering of suitable corneal endothelial graft alternatives, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as Descemet’s Stripping Endothelial Keratoplasty (DSEK) and Descemet’s Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types were also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. A total of 20 donor corneas consisting of 10 single donor corneas and 5 paired donor corneas were used in this study. Donor age ranged from 19 - 76. This RNA-seq study included 15 pooled corneas (5 each) used form CEC old, CEC young and stroma samples.
Project description:While the mouse cornea has been well characterized morphologically, the transcriptional changes have not been described in detail. To characterize the genes, pathways, and transcriptional regulators involved in mouse cornea development and aging, we isolated whole cornea from wildtype CB6 mice at several developmental timepoints and every 6 months in the adult. Corneal epithelium and stroma were isolated at one timepoint to provide insights into the genes that are unique to each tissue. Total RNA was purified from whole mouse cornea at 12 time points over the life of the mouse and from corneal epithelium and stroma at a single timepoint.
Project description:Expression of microRNAs (miRNA) was examined in the wild type mouse cornea at postnatal day 9 (PN9) before stratification of the corneal epithelium and at 6 weeks of age when the epithelium is fully stratified. Using Sanger miRBase Version 10.0, we analysed 568 miRNAs across 4 biological replicates of the developing (PN9) and mature (6-week-old) mouse cornea.
Project description:To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea.
Project description:Human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo,we tried to activate proliferation of HCEC by inhibiting cyclin-dependent kinase inhibitors.We have here demonstrated microarray data of transduced human corneal endothelial cell lines. Affymetrix human U133 plus 2.0 array was used to transcriptionally profile to compare cultured human corneal endothelial cells and transduced human corneal endothelial cells.
Project description:Purpose: Klf5 plays a critical role in the mouse ocular surface (Kenchegowda et al., 2011. Dev Biol. 356:5-18). Here, we compare wild-type (WT) and Klf5-conditional null (Klf5CN) corneal gene expression at postnatal day-11 (PN11) and PN56 to identify the Klf5-target genes. Methods: Gene expression was compared using Affymetrix microarrays with QPCR validation. Transient transfection assays examined the effect of Klf5 on selected target gene promoter activities. Whole-mount corneal immunofluorescent staining examined neovascularization and CD45+ macrophage influx. Results: Expression of 714 and 753 genes was increased, and 299 and 210 genes decreased in PN11 and PN56 Klf5CN corneas, respectively, with 366 concordant increases, 72 concordant decreases and 3 discordant changes. Canonical pathway analysis identified 35 and 34 significantly (p<0.001) enriched pathways at PN11 and PN56, respectively, with 24 common pathways. PN56 Klf5CN corneas shared 327 increases and 91 decreases with the previously described Klf4CN corneas (Swamynathan et al., 2008. IOVS 49:3360-70). Angiogenesis and immune response-related genes were affected consistent with lymphangiogenesis and macrophage influx in Klf5CN corneas, respectively. Expression of 1574 genes was increased and 1915 decreased, in the WT PN56 compared with PN11 corneas. Expression of many collagens, matrix metalloproteinases and other extracellular matrix associated genes decreased in WT corneas between PN11 and PN56, while that of solute carrier family members increased. Conclusions: Differences in PN11 and PN56 corneal Klf5-target genes reveal dynamic changes in Klf5 functions during corneal maturation. Klf4- and Klf5-target genes do not overlap, consistent with their non-redundant roles in the mouse cornea. Wild type and Klf5-conditional null mouse corneal gene expression at postnatal day-11 and -56 was compared by Affymetrix mouse whole genome 430 2 arrays. Four age-matched PN56 WT and Klf5CN mice, and 3 age-matched PN11 WT and Klf5CN mice each were used for comparison of corneal gene expression by microarrays. Two dissected corneas from each mouse were pooled for isolation of total RNA using the RNeasy Mini kit (Qiagen, Germantown, MD). The quality and integrity of the isolated total RNA was confirmed using an Agilent Bioanalyzer. Total RNAs were amplified and labeled using a 3' IVT Express Kit (Affymetrix Inc., Santa Clara, CA), and hybridized to Affymetrix MG 430 2 chips following the protocol suggested by the manufacturer.
Project description:Integrity of the cornea, the most anterior part of the eye is indispensable for vision. 45 million individuals are bilaterally blind and another 135 millions have severely impaired vision in both eyes because of loss of corneal transparency; treatments range from local medications to corneal transplants and more recently to stem cell therapy. The corneal epithelium is a squamous epithelium that is constantly renewing with a vertical turnover of seven to fourteen days in many mammals3. Identification of slow cycling cells (label-retaining cells or LRCs) in the limbus of the mouse has led to the notion that the limbus is the niche for the stem cells responsible for the long-term renewal of the cornea4; hence, the corneal epithelium is supposedly renewed by cells generated at and migrating from the limbus, in striking opposition to other squamous epithelia in which each resident stem cell has in charge a limited area of epithelium. Here, we show that the corneal epithelium of the mouse can be serially transplanted, is self-maintained and contains oligopotent stem cells with the capacity to generate goblet cells if provided with a conjunctival environment. In addition, the entire ocular surface of the pig, including the cornea, contains oligopotent stem cells (holoclones) with the capacity to generate individual colonies of corneal and conjunctival cells; hence, the limbus is not the only niche for corneal stem cells and corneal renewal is not different from other squamous epithelia. Experiment Overall Design: Expression profile difference between keratinocyte clones from conjunctiva and cornea (3 individual clones in each group)