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Y. Hayashida, W. Li, H. He, Y. Matsumoto, S.C. G. Tseng; Isolation and Demonstration of Limbal Epithelial Progenitor Cells Deep in the Limbal Stroma . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3020.
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© ARVO (1962-2015); The Authors (2016-present)
To demonstrate that limbal epithelial stem cells might be situated much deeper into the limbal stroma so that they cannot be isolated in total by conventional dispase digestion.
Human limbal corneal epithelial sheets were isolated by 10 mg/ml dispase II in KSFM at 37 °C for 2h from the corneoscleral ring after corneal transplantation, trypsinized, and cultured on plastic with or without 3T3 fibroblast feeder layers in SHEM medium. The remaining limbal stroma was surgically dissected and digested by 2 mg/ml collagenase A solution in serum–free KSFM medium at 37 °C for 16h to isolate embedded cells, which were then cultured on plastic with or without 3T3 fibroblast feeder layers in KSFM. Clonal growth was monitored daily under a phase contrast microscope and the epithelial phenotype was verified by immunostaining to pancytokeratins using limbal cryosections as a control.
Dispase–isolated limbal epithelial sheets generated monolayers of large squamous epithelial cells on plastic in SHEM, but small epithelial colonies on 3T3 feeder layers in either SHEM at day 6, and reached confluence at day 8. In contrast, collagenase–isolated stromal cells contained clusters of pancytokeratin–positive small epithelial cells when seeded on plastic without 3T3 feeder layers. They could generate large epithelial colonies consisting of small epithelial cells on 3T3 feeder layers in KSFM as early as day 2, and reached confluence at day 5.
These findings indicate that limbal epithelial progenitor cells lie deeper in the limbal stroma than expected, and these cells cannot be isolated in total by conventional dispase digestion, and further suggest that modifications are needed to achieve effective ex vivo expansion.
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