Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadetermined with various assays.Results: Treatment with TSA and the Nrf2-ARE activator resulted inincreased inhibition of the TGF-β-induced myofibroblastdifferentiation as compared with treatment with DPI or NAC.Furthermore, TSA also decreased cellular ROS and H2O2accumulation induced by TGF-β, whereas it elevated intracellularGSH level and cellular total antioxidant capacity. In addition, TSAinduced Nrf2 nuclear translocation and up-regulated the expression ofNrf2-ARE downstream antioxidant genes, whereas Nrf2 knockdownby RNA interference blocked the inhibition of TSA on myofibroblastdifferentiation.Conclusions: This study provides the first evidence implicating thatTSA inihibits TGF-β-induced ROS accumulation and myofibroblastdifferentiation via enhanced Nrf2-ARE signaling.Commercial Relationships: Qingjun Zhou, None; Lingling Yang,None; Mingli Qu, NoneSupport: National Natural Science Foundation of China 81170816Program Number: 5250 Poster Board Number: C0169Presentation Time: 2:45 PM - 4:30 PMExploring cell plasticity: the corneal keratocyte and beyondCarol A. Greene, Trevor Sherwin, Colin R. Green. Ophthalmology,University of Auckland, Auckland, New Zealand.Purpose: Corneal keratocytes have a remarkable ability to healwounds in the cornea throughout life and also exhibit a high level ofcell plasticity. However, their differentiative potential is not limitedto repair phenotypes. It has been shown in our laboratory that it ispossible to induce human and rat stromal keratocytes to expressneuron specific proteins by adding specific growth factors to theculture medium. The purpose of this study was to carry out ex vivoand in vivo experiments in an effort to uncover the extent of cornealkeratocyte plasticity. Furthermore, to explore and compare thepotential of other cell types to exhibit similar cell plasticity.Methods: Tissue and cell culture was used for in vitro experiments.Immunohistochemistry and RT PCR were used to investigate proteinand gene expression respectively. Confocal laser scanningmicroscopy was used for imaging cells and tissue slices.Results: Keratocytes obtained from adult humans and rats expressedNestin, NF-200, and MAP2 when cultured in a neuronaldifferentiation medium containing EGF, FGF and IGF-1. RT PCRrevealed up regulation of GAD1, SYN1, SOX2, SOX10 andNOTCH1. Subsequent in vivo studies also confirmed the expressionof Nestin, MASH1, and NF200. Adult rat xiphosternum derivedchondrocytes cultured in the same neurogenic media expressed NF-200, MAP2, GAP43 and β-III-tubulin. Adult human cornealkeratocytes when cultured for 3 weeks in a chondrogenicdifferentiation medium containing TGF-β3 and dexamethasoneformed spheres that produced Collagen Type II.Conclusions: Apart from producing neuron specific proteins such asNF-200 and MAP2, the genes SOX2, SOX10 and NOTCH1 whichare associated with neurogenesis were upregulated in corneal slicescultured in the neurogenic medium. Also, the expression of genessuch as GAD1 and SYN1 which are associated with neurotransmittersynthesis and synapse formation indicate that the neuronal cellsproduced might be functional. Similar to corneal stroma, cartilagefrom adult rats contained cells that acquire a neuronal phenotype andexpress neuron specific proteins. Corneal keratocytes also have thepotential to produce the cartilage specific protein Collagen II in cellculture and ex vivo slice culture when exposed to conditions whichfavour chondrogenic differentiation. These findings will have animpact on aspects of tissue regeneration research as well as ourcurrent understanding of adult cell plasticity.Commercial Relationships: Carol A. Greene, None; TrevorSherwin, None; Colin R. Green, NoneProgram Number: 5251 Poster Board Number: C0170Presentation Time: 2:45 PM - 4:30 PMReprogramming Genes Are Expressed during SpheroidalCulture of Corneal Stromal CellsYong-Soo Byun, Lisette Yco, Brittany Shaheen, Abhishek Sharma,Shweta Chaudhary, Sonal Gandhi, Sarmad H. Jassim, Joy Sarkar,Sapna Tibrewal, Sandeep Jain. Ophthalmology and Visual Sciences,University of Illinois at Chicago, Chicago, IL.Purpose: Corneal stromal cells transform to precursor cells inspheroidal culture. We determined whether reprogramming genes areexpressed during this transformation.Methods: Stromal cells were isolated from murine corneas bysequential collagenase digestion and adherent culture to generate apopulation of mixed stromal cell phenotypes. After trypsinization,spheroidal culture was performed by seeding dissociated stromal cellsonto ultra-low attachment plates containing serum-free mesenchymalstem cell culture medium. Spheroids were analyzed for expression ofreprogramming genes. Spheroids in culture were induced todifferentiate to keratocytes, fibroblasts, myofibroblasts, neural cells,adipocytes, and osteocytes using the appropriate medium.Results: Sphere formation occurred in ultra-low attachment platesbut not in adherent culture of stromal cells. Of the essentialreprogramming genes, Oct4 and Sox2 were upregulated in thespheroids, but not c-Myc or Klf4. Myc genes were differentiallyregulated; c-Myc was downregulated and N-Myc was upregulated.Forced differentiation of spheroids reverted them to keratocytes,fibroblasts, and myofibroblasts and transformed them to neural cellsbut not to adipocytes or osteocytes.Conclusions: In spheroidal culture, adherent corneal stromal cellstransform to a less differentiated precursor form, possibly due toupregulation of essential reprogramming genes. This finding suggeststhat terminally differentiated stromal cells possess inherent plasticityand multilineage potential that can be activated.Commercial Relationships: Yong-Soo Byun, None; Lisette Yco,None; Brittany Shaheen, None; Abhishek Sharma, None; ShwetaChaudhary, None; Sonal Gandhi, None; Sarmad H. Jassim, None;Joy Sarkar, None; Sapna Tibrewal, None; Sandeep Jain,PCT/US20/51562 (P)Support: NIH Grant EY018874Program Number: 5252 Poster Board Number: C0171Presentation Time: 2:45 PM - 4:30 PMTissue-engineered corneal stromal substitutes transplanted in theliving feline model: biocompatibility, ultrastructure andperformanceMarie Boulze Pankert 1, 2 , Benjamin Goyer 3 , Myriam Bareille 2 ,Kanwarpal Singh 2 , Stephanie Proulx 3, 5 , Isabelle Brunette 2, 4 .1 Departement d'ophtalmologie, Centre Hospitalier Universitaire del'Université de la Mediterranée, Marseille, France; 2 Maisonneuve-Rosemont Hospital Research Center, Montreal, QC, Canada; 3 CentreLOEX de l’Université Laval, Génie tissulaire et régénération - Centrede recherche FRQS du Centre Hospitalier Universitaire, Quebec, QC,Canada; 4 Department of Ophthalmology, University of Montreal,Montreal, QC, Canada; 5 Département d’ophtalmologie et d’otorhino-laryngologie,Faculté de médecine, Université Laval, Quebec,QC, Canada.Purpose: Tissue engineering of a corneal stroma is a promisingalternative to overcome the limitations of lamellar cornealreplacement with eye bank human corneas. We have demonstratedthe feasibility of engineering all three corneal layers. The aim of this©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.