A Novel Isolation Technique Of Progenitor Cells In Human Corneal Epithelium Using Uncoated Dishes

¹ From the Department of Corneal Tissue Regeneration, Tokyo University Graduate School of Medicine, Tokyo, Japan.
² Life Science Laboratory, Shimadzu Corporation, Division of Advanced Clinical Proteomics, Institute of Medical Science, University of Tokyo.
³ Department of Ophthalmology, Tokyo University Graduate School of Medicine, Tokyo, Japan.
⁴ Department of Microbiology and Immunology, Keio University School of Medicine.

^* To whom correspondence should be addressed. E-mail: syamagami-tky{at}umin.ac.jp.

	Abstract

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 show that a novel isolation technique using uncoated dishes and floating culture can 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 uncoated 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 uncoated 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.