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S. Yamagami, S. Yokoo, T. Usui, S. Amano, M. Araie, J. Hamuro; A Novel Isolation Technique of Limbal Epithelial Progenitor Cells Using Uncoated Dishes. Invest. Ophthalmol. Vis. Sci. 2008;49(13):6063. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The existence of limbal epithelial progenitor cells has long been suggested, but progenitor cells have not yet been isolated. We show a novel isolation technique to enrich progenitor cells using uncoated dishes.
Human corneal limbal tissues were digested by collagenase and completely disaggregated single cells were cultured overnight in serum- and feeder cell-free medium containing B27 and 20ng/ml epidermal growth factor in uncoated dishes. Marker expressions on the adherent cells were investigated. Extra-cellular matrix on the adherent cells was identified by mass spectrometer.
Adherent single cells from limbal epithelium formed round colonies and 20% of the colonies reconstituted a complete three-dimensional human corneal epithelial equivalent from single cells in serum-, feeder- and bovine pituitary extract-free medium. The colonies expressed P63, nestin, and microtubule-associated protein 2. Overlayered cells on the basal cells expressed cytokeratin3 and cytokeratin12. This technique was also applied to the isolation of other undifferentiated human epithelial cells. Laminin-5 was detected by mass spectrometric analysis. Hydrophilic tubes (Sumilon Stem Full®, Sumitomo Bakelite Co., Ltd.) to protect cell-adhesion to the inside of the tubes (used for cell collection throughout this study) are essential for efficient isolation of adherent epithelial progenitor cells expressing laminin-5.
These findings indicate that our new technique using uncoated dishes allows the isolation of limbal epithelial progenitor cells from human cornea and that laminin-5 has a critical role in the adhesion of these cells. This isolation technique may deserve wider attention in the field of squamous cell biology.
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