A novel isolation technique of progenitor cells in human corneal epithelium using non-tissue culture dishes.

The existence of adult stem cells or progenitor cells in the human corneal epithelium (i.e., self-renewing squamous cells) has long been suggested, but these cells have not yet been isolated. Here we describe a novel isolation technique using non-tissue culture dishes to enrich progenitor cells, which are able to reconstitute a three-dimensional human corneal epithelial equivalent from single cells in serum-, feeder-, and bovine pituitary extract-free medium. These cells showed original tissue-committed differentiation, a high proliferative capacity, and limited self-renewal. Laminin-5 was measured by mass spectrometric analysis. Pretreatment of cells with anti-laminin-5 antibody demonstrated that laminin-5 was important in allowing corneal epithelial progenitor cells to adhere to non-tissue culture dishes. Hydrophilic tubes (used for cell collection throughout this study) are essential for efficient isolation of adherent corneal epithelial progenitor cells expressing laminin-5. These findings indicate that our new technique using non-tissue culture dishes allows the isolation of progenitor cells from human corneal limbal epithelium and that laminin-5 has a critical role in the adhesion of these cells.