Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Corneadisplaying the characteristics of photoreceptors. Here, we haveexamined the hypothesis that retinal pigment epithelial (RPE) cellscan be produced from primary human limbal cultures.Methods: Human corneoscleral rims were incubated withcollagenase to facilitate the removal of the limbal epithelium (LE).LE was dissociated non-enzymatically and cultured in the presence ofEGF, FGF2 and Noggin to produce floating neurospheres (LiNS).LiNS were plated on a geltrex matrix to form adherent colonies. Inparallel, primary limbal cells were cultured in keratinocyte media toproduce adherent LE cell monolayers. Primary LE and LiNS cultureswere examined by microscopy and immunocytochemical analysis.Results: Two different populations of neurospheres were evident inprimary human LiNS cultures; pigmented and non-pigmented.Comparison of primary keratinocyte and neurosphere culturesrevealed the predominance of non-pigmented LiNS in cultures withstromal keratocyte contamination. Pigmented LiNS expressed theocular transcription factors PAX6, OTX2 and MITF, and containedcells expressing the RPE specific markers RPE65 and ZO1.Conclusions: Culture of human limbal epithelial stem cells in thepresence of EGF, FGF2 and Noggin leads to the induction of LiNScontaining pigmented cells expressing the RPE cell markers MITF,RPE65 and ZOI.Human limbal neurosphereglycosaminoglycans (GAGs). MPS are progressive disorders inwhich GAGs and their metabolic derivatives accumulate inlysosomes compromising cellular activity and ultimately leading tocell death. MPS VII, Sly syndrome, caused by a mutation in β-glucuronidase, manifests as hepatomegaly, skeletal dysplasia, shortstature, corneal clouding and developmental delay, due to theaccumulation of heparan sulfate (HS), dermatan sulfate andchondroitin 4,6-sulfate (CS). Current treatment regimens for MPS arenot effective for treating corneal clouding and mental development.Methods: We hypothesized that umbilical mesenchymal stem cells(UMSC) transplanted into the corneal stroma can participate in thecatabolism of GAGs, thus providing a means of cell therapy for MPS.For such, human UMSC were intrastromally transplanted intocorneas of 1, 2 and 3 month-old MPS VII mice.Results: UMSC transplantation restored the dendritic and hexagonalmorphology of host keratocytes and endothelial cells, respectively,and in vivo confocal microscopy (HRTII) revealed reduced cornealhaze. Immunohistochemistry using antibodies against HS and CSchains, as well as, LAMP2 revealed a decrease in GAG content andboth lysosomal number and size in the treated corneas to levelssimilar to that of littermate controls. Labeling UMSC intracellularcompartments prior to transplantation revealed the distribution ofUMSC exosomes throughout the corneal stroma and endothelium. Anin vitro co-culture assay between skin fibroblasts isolated fromMPSVII mice and UMSC labeled with LysoSensor demonstrated thatneutral exosomes released by the UMSC are up taken by thefibroblasts and proceed to fuse with the acidic lysosomes.Conclusions: Therefore, transplanted UMSC participate inextracellular GAG turnover and aid host keratocytes to metabolizeaccumulated GAG, suggesting that UMSC could be a goodalternative for treating corneal defects associated with MPS and othercongenital metabolic disorders. Moreover, given the simplicity of thetreatment, we suggest it as prophylactic treatment upon diagnosis inorder to avoid the development of corneal clouding.Commercial Relationships: Vivien J. Coulson-Thomas, None;Bruce Caterson, Abcam, Cambridge, UK (I), CosmoBio, Japan (I);Chia-Yang Liu, None; Winston W. Kao, NoneSupport: NIH/NEI RO1 EY021768, Research to Prevent Blindness,and Ohio Lions eye Research FoundationHuman LiNS cells immunostained for RPE65 (green) and zonusoccludin-1 (red)Commercial Relationships: Samuel McLenachan, None; DanaZhang, None; Fred K. Chen, NoneProgram Number: 1009 Poster Board Number: B0314Presentation Time: 1:00 PM - 2:45 PMTranspalntation of umbilical mesenchymal stem cells cures thecorneal defects of Mucopolysaccharidosis VII miceVivien J. Coulson-Thomas 1 , Bruce Caterson 2 , Chia-Yang Liu 1 ,Winston W. Kao 1 . 1 Ophthalmology, University of Cincinnati,Cincinnati, OH; 2 Laboratory of Connective Tissue Biology, School ofBiosciences, Cardiff University, Cardiff, United Kingdom.Purpose: Mucopolysaccharidoses (MPS) are a family of relateddisorders caused by a mutation in one of the lysosomalexoglycosidases required for the sequential degradation ofProgram Number: 1010 Poster Board Number: B0315Presentation Time: 1:00 PM - 2:45 PMDental Pulp: a Source of Stem Cells with Keratocyte PotentialMartha L. Funderburgh 1 , Fatima N. Syed-Picard 2 , Charles S. Sfeir 2 ,James L. Funderburgh 1 . 1 Department of Ophthalmology, Univ ofPittsburgh Sch of Med, Pittsburgh, PA; 2 Center for CraniofacialRegeneration, University of Pittsburgh, Pittsburgh, PA.Purpose: Blindness due to corneal stromal opacity can besuccessfully treated by allografts; however, inflammation or previousgraft rejections predict poor outcome for some allogenic tissue in thestroma. These difficult cases may benefit from autologous stem celltherapy or from autologous grafts of bioengineered tissue. Dentalpulp contains a potent stem cell population with immune-suppressiveproperties, cells that could be the autologous stem cells ideal forcorneal regeneration. The purpose of this study was to explore thepotential for dental pulp stem cells (DPSC) to adopt a keratocytephenotype.Methods: Dental pulp extracted from human molars was dispersedwith collagenase, cultured and used at passage 3. Cells expressingCXCR4 protein were isolated using MACS technology. Expressionof stem cell genes was determined using flow cytometry and qPCR.Differentiation to keratocytes was carried out on collagen gels oraligned nanofiber substrata in serum-free medium containing FGF2©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. 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