Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaProgram Number: 1018 Poster Board Number: B0323Presentation Time: 1:00 PM - 2:45 PMIsolation and characterization of p75NTR positive and highproliferativecorneal endothelial cells from the human cornealendotheliumSusumu Hara, Ryuhei Hayashi, Tomofumi Kageyama, MotokazuTsujikawa, Kohji Nishida. Ophthalmology, Osaka UniversityGraduate Scool of Medicine, Suita, Japan.Purpose: The corneal endothelium is believed to be developmentallyoriginated from periocular mesenchyme via neural crest. The humancorneal endothelial progenitor cells (HCEPs) have been investigatedbecause of their potential availability for the tissue regenerativemedicine. However, the existence and the properties of HCEPs havenot been elucidated yet. We attempted to isolate the HCEPs from thehuman corneal endothelium by using the specified culture system andp75 neurotrophin receptor (p75NTR).Methods: The Descemet's membranes were stripped from the humancornea, then, treated with a cell dissociated reagent. To isolate theHCEPs, the endothelial cells were seeded on the dish coated withlaminin and cultured in the serum-free media containing basicfibroblast growth factor. Expression of neural crest markers in theisolated HCEPs was examined by real-time PCR andimmunostaining.Results: The proliferating cells were appeared at around 14 daysafter the seeding, exhibited a bipolar, spindle-shaped morphology,similar to neural crest cells. Interestingly, the proliferating cellsexpressed neural crest markers, p75NTR and Sox9. The colonyforming efficiency was approximately 0.31±0.04%, showed nosignificant relation to donor ages. The proliferating cells were able toundergo passage several times in younger donors below 60 years old,and the proliferative capability was higher than that of human cornealendothelial cells cultivated by the conventional method with fetalbovine serum containing media.Conclusions: We succeeded in the isolation of HCEPs which hadhigh p75NTR expression and proliferative capability.Commercial Relationships: Susumu Hara, None; RyuheiHayashi, None; Tomofumi Kageyama, None; MotokazuTsujikawa, Shionogi & Co. (C), Daiichi Sankyo Co. (F), DaiichiSankyo Co. (R), Santen Co. (R), AMO Co. (R); Kohji Nishida,Alcon (C), Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F),Osaka University (P)Support: Grants-in-Aid for Scientific Research from the Ministry ofHealth, Labor and Welfare and from National Institute of BiomedicalInnovation in JapanProgram Number: 1019 Poster Board Number: B0324Presentation Time: 1:00 PM - 2:45 PMSpatial transcriptome of human cornea using next generationsequencerSuguru Nakagawa 1 , Tomohiko Usui 1 , Hiroki Ueda 2 , Genta Nagae 2 ,Shogo Yamamoto 2 , Satoru Yamagami 1 , Hiroyuki Aburatani 2 , ShiroAmano 1 . 1 Ophthalmology, Univ of Tokyo Sch of Med, Tokyo, Japan;2 Div of Genome Science, RCAST, University of Tokyo, RCAST,University of Tokyo, Tokyo, Japan.Purpose: Cornea consists of anatomically distinct three layers,corneal epithelium (CEp), corneal stroma (CS), corneal endothelium(CEn), that have different developmental lineages and molecularfunctions. To reveal their transcriptional program, we performedspatial gene expression analysis of human cornea using nextgeneration sequencer.Methods: 100 ng of total RNA was extracted from themacroscopically dissected tissue fractions; CEp, CS, CEn, limbalepithelium (LEp) and conjunctiva (Cj) of the human donorcorneoscleral tissue. For each sample, approximately 150 million of100-bp, paired-end reads were sequenced (HiSeq2000, Illumina) andmapped against transcriptome database using BWA aligner. Quantityof 86,817 transcripts was corrected by gene/exon length(RPKM/FPKM), and then compared using regression analysis.Results: 24,601 Transcript (28.3%) were expressed in common withall cell fractions. Global comparison of each spatial transcriptomeshowed that CS shows the closest pattern against CEn (correlationcoefficient (R^2); CEn vs. CS 0.844, vs. CEp 0.807, vs. LEp 0.811,vs. Cj 0.821). We identified the candidate transcripts significantlyupregulated in CEn against other tissue fractions; CEp (n=1,760),LEp (n=1,513), CS (n=1,482), Cj (n=1,322), respectively. Ascommonly up-regulated transcripts in the CEn, we listed up morethan 300 coding genes in addition to previously reported CEnspecificmarkers such as COL8A2, CA2, SLC4A4, CDH2.Conclusions: We successfully demonstrated the layer-specificpattern of transcriptome and identified the novel up-regulated genesin CEn. This resource information is useful for understanding thespatial difference in transcriptional program to modulate celllineages.Commercial Relationships: Suguru Nakagawa, None; TomohikoUsui, None; Hiroki Ueda, None; Genta Nagae, None; ShogoYamamoto, None; Satoru Yamagami, None; Hiroyuki Aburatani,None; Shiro Amano, Topcon (P)Program Number: 1020 Poster Board Number: B0325Presentation Time: 1:00 PM - 2:45 PMHuman cornea proteome: Identification and quantitation of theproteins of the three main layers including epithelium, stromaand endotheliumThomas Dyrlund 1 , Ebbe Toftgaard Poulsen 1 , Carsten Scavenius 1 ,Camilla Lund Nikolajsen 1 , Ida B. Thøgersen 1 , Henrik Vorum 2 , Jan J.Enghild 1 . 1 Department of Molecular Biology, University of Aarhus,Aarhus C., Denmark; 2 Department of Ophthalmology, AalborgHospital, Aarhus University Hospital, Aalborg, Denmark.Purpose: Diseases of the cornea are common and refer to conditionslike infections, injuries and genetic defects. Morphologically, manycorneal diseases affect only certain layers of the cornea and separateanalysis of the individual layers is therefore of interest to explore thebasic molecular mechanisms involved in corneal health and disease.Methods: The three main layers including the epithelium, stroma andendothelium of healthy human corneas were isolated and the proteinswere (i) separated by SDS-PAGE followed by in-gel trypsinization,(ii) in-solution digested without prior protein separation or, (iii) insolutiondigested followed by cation exchange chromatography. Theresulting peptides were separated by LC-MS/MS and analysed on aTripleTOF 5600 mass spectrometer. Proteins were identified in theSwiss-Prot database using the Mascot algorithm and quantified usingMascot Distiller. Data extraction and processing was done using MSData Miner.Results: A total of 3250 unique Swiss-Prot annotated proteins wereidentified in human corneas, 2737 in the epithelium, 1679 in thestroma and 880 in the endothelial layer. Of these, 1787 proteins havenot previously been identified in the human cornea by massspectrometry. In total, 771 proteins were quantified, 157 based on insolutiondigestion and 770 based on SDS-PAGE separation followedby in-gel digestion of excised gel pieces. Protein analysis revealedthat many of the identified proteins were human plasma proteinsinvolved in the complement system, coagulation and defence againstpathogen infections.Conclusions: The separation of human corneas into the three mainlayers combined with modern mass spectrometry provides new©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.