May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
A Novel Isolation Technique of Limbal Epithelial Progenitor Cells Using Uncoated Dishes
Author Affiliations & Notes
  • S. Yamagami
    Univ of Tokyo Sch of Med, Bunkyo-ku, Japan
    Corneal Tissue Regeneration,
  • S. Yokoo
    Univ of Tokyo Sch of Med, Bunkyo-ku, Japan
    Corneal Tissue Regeneration,
  • T. Usui
    Univ of Tokyo Sch of Med, Bunkyo-ku, Japan
    Ophthalmology,
  • S. Amano
    Univ of Tokyo Sch of Med, Bunkyo-ku, Japan
    Ophthalmology,
  • M. Araie
    Univ of Tokyo Sch of Med, Bunkyo-ku, Japan
    Ophthalmology,
  • J. Hamuro
    Immunology, Keio Univ Sch of Med, Shinjuku-ku, Japan
  • Footnotes
    Commercial Relationships  S. Yamagami, None; S. Yokoo, None; T. Usui, None; S. Amano, None; M. Araie, None; J. Hamuro, None.
  • Footnotes
    Support  Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 6063. doi:https://doi.org/
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      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/.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: cornea: epithelium • regeneration 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×