Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Kyung-Sun Na, None; Jeewon Mok,None; Jungmook Lyu, None; Choun-Ki Joo, NoneProgram Number: 996 Poster Board Number: B0301Presentation Time: 1:00 PM - 2:45 PMHuman limbal epithelium expanded in a complex medium ormedium with human serum: a comparison of morphology andcytokeratin expressionMeeta Pathak 1 , Kristiane Haug 1 , Aboulghassem Shahdadfar 1 , EliGulliksen 1 , Magnus Roeger 3 , Liv Drolsum 1 , Jon K. Slettedal 1 , BjornNicolaissen 1 , Katerina Jirsova 2 . 1 Centre for Eye Research, Dept. ofOphthalmology, Oslo University Hospital, Oslo, Norway;2 Laboratory of the Biology and Pathology of the Eye, GeneralTeaching Hospital and Charles University, Prague, Czech Republic;3 Dept. of Pathology, Oslo University Hospital Ullevål, Oslo, Norway.Purpose: We compared aspects of ultrastructural morphology andthe expression of selected cytokeratins in human limbal epitheliumexpanded in a complex medium (COM), which contains variousrecombinant growth factors, hormones, Cholera toxin and fetalbovine serum (FBS), and culture medium with human serum (HS) asthe only growth supplement (HS).Methods: Limbal biopsies retrieved from corneo-scleral rings wereplaced with the epithelium facing down on the basement membranesurface of samples of human amniotic membranes. The biopsies werecultured in parallel in either COM or HS at 37°C, 5% CO2, and 95%air. Culture medium was changed every 2-3 days and the epitheliumwas expanded for 3 weeks. Specimens were examined by lightmicroscopy, transmission electron microscopy (TEM) andimmunohistochemistry. Expression of CKs 3, 7, 12, 14, and 19 wasevaluated using fluorescence microcopy, and intensity was graded ona scale from 0 (negative) to 3 (strongly positive).Results: By TEM, cytoplasmic density and distribution ofintermediate filaments showed a similar pattern in epithelial cellsexpanded in both types of medium. In basal cells, the filaments wereloosely organized and inconspicuous apart from those associated withintercellular junctions. Clusters of linear or wavy intermediatefilaments (tonofilaments) were encountered to a varying extent in thecytoplasm of cells in the supra-basal layers.By immunohistochemistry, expression of CK3 was not detected(grade 0) in sections of expanded epithelium regardless of type ofmedium. Both COM and HS supported the expression of CK 7, 12,14 and 19, and grading of the samples for intensity of fluorescencerevealed a similar pattern in epithelium engineered in the two typesof medium. Intensity of fluorescence in sections of epithelium labeledfor CK12 and CK19 was evaluated as weak (grade 1), while theintensity in sections of epithelium labeled for CK7 and CK 14 wasevaluated as strongly positive (grade 3).Conclusions: Our findings indicate that COM and HS may equallysupport the expression of selected CKs, suggesting similar degrees ofepithelial proliferation and differentiation. Further, a difference indensity of cytoplasmic intermediate filaments between basal andsupra-basal cells indicates a maintained gradient for epithelialdifferentiation in both types of medium.Commercial Relationships: Meeta Pathak, None; Kristiane Haug,None; Aboulghassem Shahdadfar, None; Eli Gulliksen, None;Magnus Roeger, None; Liv Drolsum, None; Jon K. Slettedal,None; Bjorn Nicolaissen, None; Katerina Jirsova, NoneProgram Number: 997 Poster Board Number: B0302Presentation Time: 1:00 PM - 2:45 PMComparative gene expression analysis of human cornea limbalepithelial stem cells and differentiated corneal epitheliumGoran Petrovski 1, 2 , Reka Albert 1, 2 , Zoltan Vereb 2 , Morten C. Moe 3 ,Ole Kristoffer Olstad 4 , Andras Berta 1 , Laszlo Fesus 2 . 1 Department ofOphthalmology, University of Debrecen, Medical and Health ScienceCenter, Debrecen, Hungary; 2 Stem Cells and Eye ResearchLaboratory, Department of Biochemistry and Molecular Biology,University of Debrecen, Medical and Health Science Center,Debrecen, Hungary; 3 Department of Ophthalmology, Oslo UniversityHospital, Oslo, Norway; 4 Department of Medical Biochemistry, OsloUniversity Hospital, Oslo, Norway.Purpose: Limbal epithelial stem cells (LESCs) are responsible forcorneal epithelium regeneration. Specific molecular markers forLESCs have not been well defined. Our goal was to find new putativemarkers for these cells and to identify associated molecular pathways.Methods: Limbal tissue explants and central corneal epithelium wereharvested from cadavers (according to the Guidelines of the HelsinkiDeclaration). The explants were cultured ex vivo and expanded intoLESCs in a human serum containing medium. Genome-widemicroarray analysis was performed using Affymetrix GeneChipHuman Gene 1.0 ST Array containing more than 28,000 genetranscripts. Functional analysis using Ingenuity software was carriedout to identify pathways and molecules specific for LESCs.Results: LESCs showed upregulated expression of 10 top molecules(flavin containing monooxygenase (FMO) 1 and 2, fibronectin 1(FN1), kallikrein (KLK) 6 and 7, transcobalamin 1 (TCN1),semaphorin 3A (SEMA3A), annexin A3 (ANXA3), V-set domaincontainingT-cell activation inhibitor 1 (VTCN1) and heat shockprotein beta-8 (HSPB8), and downregulated expression of cartilageacidic protein 1 (CRTAC1), alcohol dehydrogenase class 4 mu/sigmachain (ADH7), hepatic leukemia factor (HLF), CD36, Doublecortindomain containing 5 (DCDC5), Diacylglycerol kinase beta (DGKB),protein prune homolog 2 (PRUNE2), anoctamin 4 (ANO4), deathassociated protein-like 1 (DAPL1) and carbonic anhydrase (CA6)compared to differentiated corneal epithelium (p