Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaClinical Trial: NCT01712022Program Number: 557 Poster Board Number: B0194Presentation Time: 10:30 AM - 12:15 PMBone Marrow derived Mesenchymal Stem Cells forReconstructing the Limbal NicheAli R. Djalilian 1 , Behrad Y. Milani 1 , Hossein M. Sagha 1 , PeimanHematti 2 . 1 Ophthalmology, Univ of Illinois at Chicago, Chicago, IL;2 Medicine, University of Wisconsin, Madison, WI.Purpose: In patients with limbal stem cell deficiency, in addition tothe loss of the limbal epithelial stem cells, the limbal niche isinvariably damaged/destroyed. Therefore, strategies are needed toreconstruct the limbal niche. Mesenchymal type cells have beenshown to be an important part of the niche. In this study, weexamined whether mesenchymal stem cells (MSC) from the bonemarrow (BM) can function as niche support cells.Methods: Human bone marrow MSCs were cultured in serumcontaining media. Primary human corneal epithelial cells were grownin KSFM before being passaged onto plates with BM-MSC. Colonyforming efficiency of epithelial cells co-cultured with BM-MSC wasexamined. Conditioned media from BM-MSC and limbal fibroblastswere compared in terms of their ability to promote epithelial growth.BM-MSC were also grown in decellularized human corneas toexamine their growth and differentiation in 3D.Results: As reported by others, BM-MSC supported colonyformation of corneal epithelial cells, which appeared to be dependenton the passage number of the BM-MSC. Pre-treatment of BM-MSCwith mitomycin C reduced their ability to support corneal epithelialcell growth as evident by the effect of conditioned media (non-MMCvs MMC treated) on corneal epithelial cells. BM-MSC proliferated in3D culture conditions in decellularized human cornea adopting akeratocyte-like phenotype.Conclusions: These results indicate that BM-MSC can support theclonal growth of corneal epithelial cells. More studies are needed todetermine the optimal growth conditions for their use as limbal nichecells.Commercial Relationships: Ali R. Djalilian, None; Behrad Y.Milani, None; Hossein M. Sagha, None; Peiman Hematti, NoneSupport: National Eye Institute of the National Institutes of Healthwith Career Development Grant K08EY017561-A1 (ARD) and CoreGrant EY01792, the Cless Family Foundation, and a CareerDevelopmental Award (ARD) and unrestricted departmental grantfrom Research to Prevent Blindness.Program Number: 558 Poster Board Number: B0195Presentation Time: 10:30 AM - 12:15 PMDecreased Phosphorylation at the Serine-2 Residue of the RNAPolymerase II in Corneal Epithelial Stem CellsSatoshi Kawasaki 1 , Katsuhiko Shinomiya 1 , Keita Aoi 2, 1 , ShigeruKinoshita 1 . 1 Ophthalmology, Kyoto Prefectural Univ of Med, Kyoto,Japan; 2 Faculty of Life and Medical Sciences, Doshisha University,Kyotanabe, Japan.Purpose: Continuous regeneration of stem cells is known to beessential for maintaining the life-long homeostasis of any types ofcells in the body of animals. In the stem cells of ocular surfaceepithelia, several positive and negative markers have been reported.Recent studies have shown that stem cells are adapted to hypoxicconditions with slow cell cycling, low transcriptional activity, lowermitochondrial respiration, and higher glycolysis for the generation ofadenosine-5'-triphosphate (ATP). The purpose of this present studywas to report a new marker for corneal epithelial stem cells based onlow transcriptional activity.Methods: Corneal tissue samples obtained from humans, rabbits, andmice were cryo-embedded, sliced into thin sections, fixed, andimmunostained with antibodies against the phosphorylated serine-2of the RNA polymerase II (RNAP II), a marker of activatedtranscription, as well as several positive or negative markers ofcorneal epithelial stem cells such as N-cadherin, p63, and keratin 12.Results: In all of the tissue samples, a decreased level ofphosphorylation at the serine-2 residue of the RNAP II was found atthe basal cells of the limbal epithelium. Immunostaining withantibodies against the above-described established markers forcorneal epithelial stem cells proved that those cells were, in fact,corneal epithelial stem cells.Conclusions: The findings of this study show that phosphorylatedserine-2 of the RNAP II is a new negative marker for cornealepithelial stem cells. Corneal epithelial stem cells may have a generalfeature of stem cells in terms of low transcriptional activity.Commercial Relationships: Satoshi Kawasaki, None; KatsuhikoShinomiya, None; Keita Aoi, None; Shigeru Kinoshita, SenjuPharmaceutical Co (P), Santen Pharmaceutical Co (P), OtsukaPharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)Support: 24592675Program Number: 559 Poster Board Number: B0196Presentation Time: 10:30 AM - 12:15 PMEffect of Specular Focal Distance on Endothelial Cell CountingAccuracyJackie Hai, Vivian Xue. HAI Laboratories, Inc., Lexington, MA.Purpose: To quantify the relationship between specular focaldistance and image distortion, in order to determine a predictivemodel of cell counting error based on amount of deviance from trueendothelial cell shape and size.Methods: High resolution specular image of a standard calibrationlens was captured in focus to establish baseline pachymetry of 0μm.Subsequent images captured 30μm, 40μm, 50μm, 60μm, 70μm,80μm, 90μm and 100μm from the baseline were analyzed for changesin area caused by optical distortion. Analysis was carried out in asingle-blind trial with 10 sample counts performed for each specularimage to determine mean areas.Results: Given a true area of 10,000μm 2 for the baseline distance of0μm, a negative correlation was found to exist between focal distance(F) and mean area (A). For F=30μm, A=9666μm 2 ; F=40μm,A=9444μm 2 ; F=50μm, A=9180μm 2 ; F=60μm, A=9055μm 2 ; F=70μm,A=8869μm 2 ; F=80μm, A=8767μm 2; F=90μm, A=8694μm 2 ;F=100μm, A=8467μm 2 . Using simple linear regression, the celldensity error (E) can be extrapolated based on deviance from truearea. For F=10μm, E=1.26%; F=20μm, E=2.90%; F=30μm,E=4.58%; F=40μm, E=6.53%; F=50μm, E=8.12%; F=60μm,E=9.99%; F=70μm, E=11.92%; F=80μm, E=13.91%; F=90μm,E=15.98%; F=100μm, E=18.13%.Conclusions: Cell counting error can be considered negligible at 0-29μm, non-negligible at 30-59μm and problematic at 60-100μm offocal deviance.©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.