Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaSaghizadeh 1 . 1 Regenerative Medicine Institute, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2 Ophthalmology and Cell Biology, StateUniversity of New York, Downstate Medical Center, Brooklyn, NewYork, NY; 3 University of California, Los Angeles, CA.Purpose: Human amniotic membrane (HAM) is a commonsubstratum for culturing limbal cells for future transplantation. Bestresults are obtained when amniotic epithelium is removed fromHAM. Most existing methods require long treatments and leave manycells attached to HAM. The purpose was to develop a reliable methodof HAM denuding with effective and fast removal of amnioticepithelium.Methods: Fresh HAM was mechanically separated from chorion, andcryopreserved in PBS with 10% DMSO. After thawing and washing,HAM was de-epithelialized by different methods: soaking in 0.02%EDTA in PBS at 37°C for 1 hour; EDTA followed by gentle scrapingwith electric toothbrush; EDTA followed by scraping with n-heptanolsoaked cotton tip; 125 μg/ml thermolysin in PBS at 37°C for 9 minfollowed by gentle mechanical scraping. Alternatively, HAM placedin CellCrownTM inserts was rubbed on the epithelial side for 10-30seconds with cotton tip soaked in 0.5 M NaOH and immediatelywashed in PBS. Denuded OCT-embedded HAM was cryosectionedand immunostained for typical components of limbal basementmembrane (BM), including laminin α2, γ1, and γ3 chains, α1/α2 typeIV collagen, perlecan, nidogen-2, and fibronectin. NaOH-denudedHAM was used to culture human telomerase-immortalized cornealepithelial cells, limbal cells from corneoscleral rims, and inducedpluripotent stem cells (iPSC) derived from limbal epithelial cells.Cultured cells were checked for putative stem cell marker expression(ΔNp63α, ABCG2, and keratins 14, 15, 17, and 19) byimmunostaining.Results: Control HAM was positive for all BM markers except forlaminin α2 chain that gave weak and inconsistent staining. HAM decellularizationwith EDTA or EDTA with n-heptanol left out manyadherent epithelial cells; rubbing with toothbrush produced localtears. Thermolysin and NaOH resulted in the best cell removal withcontinuous staining for all BM markers tested. However,thermolysin-treated HAM became fragile and could be easilydamaged during manipulation, which was not seen after NaOHdenuding. Corneal epithelial cell line, limbal cells, and limbalderivediPSC all grew well on NaOH-denuded HAM. Cultured cells,especially limbal cells were positive for putative stem cell markers.Conclusions: HAM de-cellularization with NaOH results in rapidand thorough amniotic cell removal, and ensures excellentpreservation of HAM structure and robust stem cell growth ondenuded HAM.Commercial Relationships: Alexander V. Ljubimov, None; DhruvSareen, None; Loren Ornelas, None; Anais Sahabian, None;David M. Hemmati, None; Chantelle A. Ghiam, None; William J.Brunken, None; Yaron S. Rabinowitz, None; Clive Svendsen,None; Mehrnoosh Saghizadeh, NoneSupport: NIH EY13431, CTSI grant UL 1RR033176, andRegenerative Medicine Institute grants.Program Number: 999 Poster Board Number: B0304Presentation Time: 1:00 PM - 2:45 PMExpansion of Human Corneal Epithelial Stem/Progenitor Cells inFeeder-Free Explant CulturesSophie X. Deng, Martin N. Nakatsu, SHEYLA GONZALEZ, Hua Mei.Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA.Purpose: To present a feeder-free culturing method of humancorneal epithelial stem/progenitor cells in vitro.Methods: Primary limbal epithelial cell (LEC) sheets isolated fromhuman sclerocorneal tissues were trypsinized to produce single LECsand co-cultured with growth arrested 3T3-J2 feeder cells for 14 days.Additionally, the outgrowth of LECs from limbal explant pieces wasalso cultured for 14 days in the presence or absence of 3T3-J2 feeder.The phenotype of the cultured LECs was assessed by their mRNAexpression level of putative stem cell markers and differentiationmarker by qRT-PCR and immunocytochemistry. The percentage ofp63 bright cells in each culture was assessed, and the cellproliferation was evaluated by the Ki67 expression and cell number.Results: We observed no significant difference in cultured LECmorphology among each culturing method. The LEC growth rateincreased over 9-fold in NF explant cultures compared to 3T3-J2explant cultures and the adjusted growth rate between NF culturesand 3T3-J2 single LEC cultures had similar yields (p>0.05). Geneexpression of putative limbal stem cell markers, ABCG2 and ΔNp63were elevated among NF cultures compared to the gold standard andexplants on 3T3-J2. We observed a 7.6-fold and 2.2-fold increase inABCG2 and ΔNp63 expression respectively when comparing NFexplant cultures to the gold standard, while there was only a 4.4-foldand 1.4-fold increase in ABCG2 and ΔNp63 expression respectivelywhen comparing 3T3-J2 explant cultures to the gold standard.However, we did observe an increase in K12 expression in NFexplant cultures when compared to the gold standard (6.2-fold).There was a large increase in Ki67 proliferation (4.0-fold) whencompared to the gold standard. There was no difference in Ki67expression between 3T3-J2 explant cultures and the gold standard.Finally, examination of p63α expression in each condition reviewedno discernable differences in the percentage of p63α bright cellsbetween the gold standard (9.0%) and NF explant cultures (10.0%),but we did see a decrease in the 3T3-J2 explant cultures (6%).Conclusions: 3T3 feeder cells may not be necessary for the growthof stem/progenitor cell population in the primary explant culture. Theexplants themselves may already contain niche factors that arerequired for the viability of corneal stem cells.Commercial Relationships: Sophie X. Deng, None; Martin N.Nakatsu, None; SHEYLA GONZALEZ, None; Hua Mei, NoneSupport: CIRM Grant TR2-01768, NIH Grant EY021797Program Number: 1000 Poster Board Number: B0305Presentation Time: 1:00 PM - 2:45 PMSox9, a determinant of hair follicle stemness, identifies a subset ofmurine basal corneal epithelial cells expressing putative stem cellmarkersRachel Sartaj 1 , Aihong Liu 1 , Elaine Fuchs 2 , Mark Rosenblatt 1 .1 Ophthalmology Department, Weill Cornell Medical College, NewYork, NY; 2 Laboratory of Mammalian Cell Biology andDevelopment, Rockefeller University, New York, NY.Purpose: To localize the expression of Sox9 in the cornea and toidentify genes co-expressed with this determinant of hair folliclestemness.Methods: Antibodies against Sox9 were used to immunostaincorneal sections from wild-type mice, and the nuclear localization ofSox9 determined by wide field and confocal microscopy.Localization of Sox9 within the cornea epithelium was confirmed byimmunostaining for GFP in Sox9-eGFP mice expressing GFP underthe control of the sox9 promoter region. Corneal epithelial cells wereisolated from Sox9-egfp mice via sequential enzymatic treatments,and the cell suspension used for FACS sorting to obtain purifiedpopulation of Sox9-expressing (GFP +) cells and Sox9 nonexpressing(GFP-) cells. RNA was separately isolated from GFP +and GFP - populations and qRT-PCR performed to determine therelative expression of putative corneal epithelial stem cell markersABCG2, p63 and N-cadherin (Cdh2) as well as the differentiationmarkers keratin-12 (Krt12) and involucrin (Ivl).©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.