title: Development of transplantable genetically modified corneal epithelial cell sheets for gene therapy.
abstract: The purpose of this study was to establish a method for the fabrication of exogenous gene-transferred, transplantable corneal epithelial cell sheets. Corneo-limbal epithelial cells collected from USA eye bank eyes were transduced with an EGFP-expressing lentiviral vector at differential MOI. Multi-layered corneal epithelial cell sheets were fabricated by co-cultivation of transduced cells and mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes. These cultured epithelial cells could be harvested as intact sheets by simply lowering the temperature. The number of EGFP-positive cells was increased as the MOI raised, and at an MOI of 100, nearly 100% of the superficial cells showed strong EGFP expression. Histological analysis revealed that EGFP was expressed in all layers of the cell sheet of which cell source was transduced with the lentiviral vector at an MOI of 100. Immunofluorescence data showed that p63 was also expressed in the basal layer of the same cell sheet. These results suggest that this technique will likely be applicable to ex vivo gene therapies for various corneal disorders.
authors.affiliation: Department of Ophthalmology, Osaka University Medical School, Room E7, 2-2 Yamadaoka, Suita, Osaka 565 0871, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Watanabe
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yamato
authors.forename: Yasutaka
authors.initials: Y
authors.lastname: Hayashida
authors.forename: Joseph
authors.initials: J
authors.lastname: Yang
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kikuchi
authors.forename: Teruo
authors.initials: T
authors.lastname: Okano
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Tano
authors.forename: Kohji
authors.initials: K
authors.lastname: Nishida
mesh_terms: D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism* / Q000637:transplantation*; D019573:Epithelium, Corneal / Q000166:cytology* / Q000637:transplantation; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans