Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaCommercial Relationships: Tingting Qian, None; Jiaxu Hong,None; JianJiang Xu, NoneProgram Number: 994 Poster Board Number: B0299Presentation Time: 1:00 PM - 2:45 PMTranscription factor screening of limbal vs. corneal epitheliumidentifies distinct patterns for SOX-9 and peroxisomeproliferator-activated receptor gamma (PPARG)Johannes Menzel-Severing, Matthias Zenkel, Friedrich E. Kruse,Ursula Schlotzer-Schrehardt. Department of Ophthalmology,University of Erlangen-Nuremberg, Erlangen, Germany.Purpose: To identify transcriptional regulators potentially involvedin limbal epithelial stem cell homeostasis and differentiation.Methods: Limbal and central corneal epithelial specimens wereobtained from four human post-mortem donor eyes using lasercapture microdissection. RNA extracted from these specimensunderwent linear amplification (MessageAmp II, Ambion). Sampleswere screened for differential expression of stem cell transcriptionfactor genes using quantitative real-time PCR arrays(SABiosciences). Candidate genes were confirmed using specificreal-time PCR hydrolysis probe assays (Roche) andimmunohistochemistry of frozen corneal sections.Results: Of 86 genes screened, nine appeared to be differentiallyexpressed, and were therefore investigated further (FOXP2,HOXA11, KLF2, SOX9, STAT3, WRN, MYC, DACH1, PPARG).Using individual assays, increased mRNA expression in limbalspecimens was confirmed for SOX9 and PPARG.Immunohistochemistry showed nuclear localization of SOX-9 inlimbal basal cells, whereas no specific staining was seen in centralcorneal epithelium. PPARG was detected within nuclei of basalepithelial cells of conjunctiva and central cornea, whileperinuclear/cytoplasmic staining was observed in small, basal cellclusters at the limbus.Conclusions: Transcription factor SOX-9 has been suggested toepithelium and the subependymal zone. Several reports haveidentified links to Wnt signaling transducer beta-catenin, for which arole in maintenance of limbal stem cells has been proposed. PPARGis a nuclear receptor associated with differentiation pathways ofadipocytes and epithelial cells as well as growth inhibition ofcarcinoma cells. Nuclear export of PPARG is mediated byMAP2K1/MEK1, which invites to speculate about a possibleupstream activity of the MAPK-pathway in putative limbalprogenitors. Further research is warranted to elucidate the functionalinvolvement of SOX-9 and PPARG in corneal epithelial celldifferentiation, and to assess the usefulness of modulating theiractivity in the context of cell therapy.Commercial Relationships: Johannes Menzel-Severing, None;Matthias Zenkel, None; Friedrich E. Kruse, None; UrsulaSchlotzer-Schrehardt, NoneProgram Number: 995 Poster Board Number: B0300Presentation Time: 1:00 PM - 2:45 PMWnt promotes proliferation of corneal epithelial progenitor cellsin xeno-feeder free cultivationKyung-Sun Na 1, 2 , Jeewon Mok 2 , Jungmook Lyu 2 , Choun-Ki Joo 1, 2 .1 Department of Ophthalmology, The Catholic University of Korea,Seoul, Republic of Korea; 2 Catholic Institutes of Visual Science, TheCatholic University of Korea, Seoul, Republic of Korea.Purpose: This study was to establish a simple, xeno-feeder freemethod for cultivating human corneal limbal explants , and also toexplore the effect of Wnt signaling on epithelial progenitor cellproliferation in this cultivation system in vitro and in vivo.Methods: The limbal tissue explants from cadaveric donor wascultured in Isocove’s Modified Dulbecco’s Medium (IMDM) and lowcalcium Panserin 801 medium in 1:1 ratio. The outgrowing cells wereexamined to characterize with flow cytometry,immunohistochemistry, and real-time PCR (RT-PCR). Sprague-Dawley male rats after alkali injuries using 1N NaOH were used forin vivo verification, after which cultivated epithelial sheets weretransplanted. Corneal opacity, re-epithelialization, andneovasculazation was observed for 2 week, and the tissue sectionsanalyzed with hematoxylin and eosin stain (HE stain). Conditionedmedia from L cells secreting Wnt-3a (Wnt3a-L-CM) was used in thecultivation system, and morphological changes and gene expressionlevel were observed.Results: There was migration of fibroblast like stromal cells fromlimbal explants initially, and then, epithelial cells migrated andgrown on stromal cells as an autofeeder layer, which was revealed bymorphological and immunohistochemical methods. RT-PCR showedthat the expression of epithelial progenitor cells are more intensecompared to fresh limbal tissue. Side population (SP) cells weredetected 0.43 ± 0.04 % (n=5) of the primary culture. Flow cytometryresulted 49.12% of E-cadherin, 40.44% of p63, and 44.55% ofABCG2 identified in the cells from explants. Maintaining cornealtransparency without neovascularization was observed in rats aftercultivated epithelial sheets transplantation for 2 weeks. Predominantincreased tightly packed epithelial cells with Wnt3a-L-CM wereobserved compare to the control CM. ABCG2, p63, Lef1, and CK3was increased in Wnt3a-L-CM.Conclusions: This explants culture system with combining media touse stromal cells as autofeeder layer, showed to expand sufficientlimbal epithelial progenitor cells in vitro and to be transplantedrestoring transparency. Also, these findings demonstrated that Wntsignaling play an important role in the proliferation of limbalepithelial progenitor in the proposed cultivation system. This studymay have clinical impact on the expansion of corneal epithelialprogenitor cells for ocular surface reconstruction.contribute to progenitor cell regulation in hair follicle, intestinal©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.