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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneaepithelium cells from fresh donor tissues were quite distinct, yetsimilar in miR expression. HCEC mRNA expression remained stableduring culture, while HCEC miR expression varied widely duringculture, thus indicating the fragility of miR expression. EMT-liketransition during culture triggered significant changes of miR traits.These findings will hopefully open a new pathway towards efficientin vitro propagation of HCECs.Commercial Relationships: Kazuko Asada, None; MunetoyoToda, None; Michio Hagiya, JCR Pharmaceuticals Co., Ltd (E);Kana Nakata, None; Morio Ueno, None; Naoki Okumura, None;Noriko Koizumi, None; Junji Hamuro, None; Shigeru Kinoshita,Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Program Number: 3228Presentation Time: 12:00 PM - 12:15 PMCXCR4 Expression Marks Cells in Limbal Stroma with thePotential to Differentiate to KeratocytesJames L. Funderburgh, Andrew Hertsenberg, Martha L.Funderburgh, Kira L. Lathrop, Yiqin Du. Ophthalmology, Univ ofPittsburgh School of Medicine, Pittsburgh, PA.Purpose: Mesenchymal cells from limbal stroma exhibit stem cellproperties and can regenerate stroma in vivo and vitro. Recentpublications showed stromal cells associating with limbal epithelialcells to express the chemokine receptor CXCR4. The study examinedthe hypothesis that stromal CXCR4 expression allows selection ofstromal stem cells with a high potential for corneal regeneration.Methods: Limbal stromal cells were recovered from human cornealrims by collagenase digestion and expanded in culture at clonaldensity in a low-serum medium containing cholera toxin(PMID:22078813). Gene expression patterns were determined byqPCR. Protein expression was assayed by flow cytometry. CXCR4+cells were isolated with MACS immunoadsorption or by FACS.Keratocyte phenotype was determined by gene and extracellularmatrix expression after culture on collagen or aligned nanofibersubstrata. Immunostaining was carried out on tissue wholemountsand sections using immunofluorescent detection with confocalmicroscopy.Results: Strong staining for CXCR4 protein was limited to smallpopulations of limbal cells subjacent to the epithelial basementmembrane. In uncultured digests and primary cultures, CXCR4positive cells amounted to one third of limbal cells in flow cytometry,but in central stroma less than 1% total cells were CXCR4+. Afterpassage, the proportion of CXCR4+ in cultured limbal cellsdecreased, particularly in confluent cultures. Separated CXCR4+cells expressed pluripotent markers Oct4, SOX2, NANOG, andneural crest marker p75NTR. Under differentiation conditions,CXCR4+ cells exhibited enhanced expression of keratocyte genesand matrix compared to CXCR4- cells.Conclusions: CXCR4 is a cell surface protein the abundance ofwhich correlates well with the potential of cells to differentiation tokeratocytes in vitro. This correlation differs from other widely knownmesenchymal stem cell markers and thereby provides a novel anduseful tool in isolating and enriching populations of stromal stemcells for use in corneal bioengineering and direct-cell based cornealtherapy.Commercial Relationships: James L. Funderburgh, None;Andrew Hertsenberg, None; Martha L. Funderburgh, None; KiraL. Lathrop, PCT/US2012/027268 (P); Yiqin Du, NoneSupport: NIH Grants EY016415, P30-EY008098, Eye & EarFoundation of Pittsburgh, Research to Prevent BlindnessProgram Number: 3229Presentation Time: 12:15 PM - 12:30 PMmiRNA EXPRESSION PROFILING IN CENTRAL ANDLIMBAL DIABETIC AND NORMAL HUMAN CORNEASUSING DEEP SEQUENCINGMehrnoosh Saghizadeh 1 , Jordan Brown 2 , Alexander V. Ljubimov 1, 3 ,Vincent A. Funari 2, 3 . 1 Surgery/Ophthalmology, Cedars-Sinai MedicalCenter, Los Angeles, CA; 2 Genomic Core, Cedars-Sinai MedicalCenter, Los Angeles, CA; 3 3David Geffen School of Medicine,University of California Los Angeles, Los Angeles, CA.Purpose: To identify and characterize miRNA expression by deepsequencing analysis in central and limbal parts of normal and diabetichuman corneas.Methods: Total RNA was extracted from age-matched humanautopsy normal (n=10) and diabetic (n=12) 8.5 mm-trephined centralcorneas and their limbi using mirVana miRNA Ambion kit. From 44total RNA samples that passed quality control standards small RNAs(22-28 bp) were gel isolated and miRNA libraries were preparedusing Illumina small RNA Sample Preparation Kit according to themanufacturer’s protocol. Samples were multiplexed and pooled intotwo lanes for a 35 bp sequencing run. Upon completion ofsequencing, FASTQ files were converted to FASTA files, andadapters were trimmed from each sequencing. FASTA files forsequencing were parsed by sample and aligned using BLAST with90% similarity to miRBase v19. Student’s t-test was performed foreach of four comparisons: normal central vs. normal limbus, normalcentral vs. diabetic central, normal limbus vs. diabetic limbus, anddiabetic central vs. diabetic limbus. For each comparison, significantmiRNAs were identified (fold change > 2, p-value < 0.05). Aknowledge base (Ingenuity Pathway Architect) was searched forbiochemical pathways that could be regulated by differentialmiRNAs.Results: A number of miRNAs with significant differential expressedin diabetic vs. normal corneas and in central vs. limbal parts werequantitatively identified; some were not present in the mirbase 19.Comparison of normal central corneas vs. normal limbi and diabeticcentral corneas vs. diabetic limbi yielded a few differential miRNAspecies. Many more miRNAs had differential expression betweennormal vs. diabetic central corneas (90) and especially, normal vs.diabetic limbi (141). In the central corneas, about equal numbers ofmiRNAs were either up- or downregulated in normal vs. diabetictissue. However, in the diabetic limbus, 133 miRNAs weredownregulated compared to normal, with only 8 upregulated.Conclusions: The results are consistent with previously observedsignificant changes in the expression of stem cell markers in diabeticlimbal epithelial cells. Abnormal miRNA expression may be animportant mechanism of diabetic changes affecting corneal epithelialstem cell compartment and wound healing. Manipulating miRNAlevels in diabetic corneas could potentially help alleviate symptomsof diabetic keratopathy.Commercial Relationships: Mehrnoosh Saghizadeh, None;Jordan Brown, None; Alexander V. Ljubimov, None; Vincent A.Funari, NoneSupport: NIH R21 EY022771, Cedars-Sinai Department of Surgery,and Regenerative Medicine Institute.Program Number: 3230Presentation Time: 12:30 PM - 12:45 PMStandardized cultivation and transplantation of limbal stem cellgrafts: Results of a phase I/II clinical trialNadia Zakaria 1, 2 , Tine Possemiers 1, 2 , Inge Leysen 1 , Jos J. Rozema 1 ,Carina Koppen 1 , Zwi Berneman 2 , Marie-Jose B. Tassignon 1 .1 Ophthalmology, Antwerp University Hospital, Antwerp, Belgium;©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.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Cornea2 Center for Cell Therapy and Regenerative Medicine, AntwerpUniversity Hospital, Antwerp, Belgium.Purpose: To describe the results of a phase I/II clinical trial forstandardized, non-xenogenic, cultivation and “no touch” surgicaltransplantation of limbal stem cell grafts.Methods: 18 eyes of 18 patients were transplanted with eitherautologous (n=15) or allogenic (n=3) limbo-amnion composite graftsthat were generated using a standardized culture protocol free ofxenogenic culture products and transplanted using a standardized “notouch” surgical technique. In vitro cellular outgrowth and phenotypeof the limbo-amnion composite graft was assessed prior totransplantation. The clinical outcome measures investigated were:corneal neovascularization, central corneal opacity, pain,photophobia and visual acuity pre and post transplantation.Results: Limbal epithelial cells showed an average outgrowth of14.2mm ±3.7mm by day 14. The majority of the cells displayed aprogenitor phenotype: p63 bright, CK14, desmoglein, ABCG2 brightand CK3/12 dim protein expression. The transplant recipients werefollowed up for a mean of 22 months (range 4-43 months). 12 out ofthe 18 transplant recipients were graded successful (12 hadanatomical success and 7 also attained some degree of functionalsuccess), giving an overall success rate of 67%. We did not see asignificant reduction in pain, photophobia or central corneal opacityfor the patient group post transplant. However, the ocular surfacephotographs for pre- and post stem cell transplantation, showed asignificant (p=0.007) reduction in the percentage area of cornealneovascularization [Fig.1].Conclusions: We have been able to show that our standardized,xenogenic free culture system and “no touch” surgical technique hasoutcome measures comparable to other clinical studies. Thistechnique has the added advantage of being free from animalcontaminants such as mouse feeder layers and foetal bovine serum.Improved functional success is attained once penetrating keratoplastyis performed following successful stem cell grafting.Program #/Board # Range: 3456-3481/D0083-D0108Organizing Section: CorneaProgram Number: 3456 Poster Board Number: D0083Presentation Time: 11:00 AM - 12:45 PMA Prospective Trial Evaluating Scleral Rebound TonometryShuchi B. Patel, Sara L. Duke, Andrew Logeman. Ophthalmology,University of Chicago, Maywood, IL.Purpose: Glaucoma is known to occur in about 75% of patientsfollowing a keratoprosthesis, but accurate pressure readings tomonitor for progression are not possible. Thus we sought todetermine if a predictable relationship exists between Goldmannapplanation tonometry (GAT) and scleral rebound tonometry (RT) toprovide an accurate and reliable assessment of intraocular pressure(IOP) via scleral measurements.Methods: A prospective non-randomized trial of individuals 18 yearsof age and older. Each had his/her IOP measured by GAT, nextcorneal RT then scleral RT on the inferotemporal sclera. Thepatient’s age, gender, refractive error, central corneal thickness(CCT), axial length (AL) and phakic status were recorded. Pearson’scorrelation and multivariate regression were used for statisticalanalysis.Results: 116 eyes from 59 patients (37-90 years old) have beenexamined to date. Mean GAT IOP was 15.91 mmHg (SD 4.13), meancorneal RT was 14.50 mmHg (SD 4.24) and mean scleral RT was48.84 (SD 21.41). Mean spherical equivalent refraction (SE) was -0.21 D (SD 2.05), mean CCT was 547.68 µm (SD 45.65), mean ALwas 24.06 mm (SD 1.21). 89 eyes were phakic and 27 werepseudophakic. Pearson analysis reveals a strong positive correlationbetween GAT and corneal RT (0.77) but weak positive correlationbetween GAT and scleral RT (0.22) as well as corneal RT and scleralRT (0.22). This trend persists when phakic and pseudophakic eyesare evaluated independently. In the final multiple regression model(Table 1) to evaluate the association of GAT, CCT, AL and SE withscleral RT, only CCT in pseudophakic eyes was found to have asignificant positive correlation (p=0.04).Conclusions: Scleral RT shows IOP measurements that areconsistently higher than corneal IOP measurements by roughly 33-34mmHg. Corneal RT correlates well to the gold standard of IOPmeasurement, GAT; unfortunately, scleral RT measurements havepoor correlation to corneal measurements independent of phakicstatus. Analysis of scleral RT with relation to GAT, CCT, AL and SEconcurrently fails to reveal a statistically significant regression modelin either phakic or pseudophakic eyes. Our study reveals that sceralRT does not provide accurate and reliable IOP measurements.Fig 1. Eyes with total limbal stem cell deficiency before (A, E) &after (C, G) limbal stem cell transplantation within the softwareprogram for corneal neovascularization (CNV) mapping: (B, F, D,H). There was a significant reduction in %area of CNV post limbalstem cell transplantation (I) (**p= 0.007), but no significant decreasein the degree of corneal opacification post stem cell transplant (J).Commercial Relationships: Nadia Zakaria, None; TinePossemiers, Aeon Astron Europe B.V. (F); Inge Leysen, None; JosJ. Rozema, None; Carina Koppen, None; Zwi Berneman, None;Marie-Jose B. Tassignon, NoneSupport: IWT-TBM 90868Clinical Trial: NCT00845117345 Corneal Surgery Non-refractive II and KeratoprosthesisTuesday, May 07, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionFinal Multivariate Regression Model Assessing the RelationshipBetween Scleral Rebound Tonometry and Ophthalmic VariablesCommercial Relationships: Shuchi B. Patel, None; Sara L. Duke,None; Andrew Logeman, None©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.
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