IOVS Journal of Pharmacology and Experimental Therapeutics
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1167/iovs.08-1987 on May 9, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:3903-3908.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-1987
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
iovs.08-1987v1
49/9/3903    most recent
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Krulova, M.
Right arrow Articles by Holan, V.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Krulova, M.
Right arrow Articles by Holan, V.

A Rapid Separation of Two Distinct Populations of Mouse Corneal Epithelial Cells with Limbal Stem Cell Characteristics by Centrifugation on Percoll Gradient

Magdalena Krulova,1,2 Katerina Pokorna,1,2 Anna Lencova,1,3 Jan Fric,1,2 Alena Zajicova,1 Martin Filipec,3 John V. Forrester,4 and Vladimir Holan1,2

1From the Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; the 2Faculty of Natural Sciences, Charles University, Prague, Czech Republic; the 3Eye Clinic Lexum, Prague, Czech Republic; and the 4Department of Ophthalmology, University of Aberdeen, Aberdeen, Scotland.

PURPOSE. To detect and isolate cells with stem cell (SC) characteristics in the limbus of the mouse.

METHODS. Limbal tissues from BALB/c mice were trypsin-dissociated and separated on the gradient Percoll (Fluka, Buchs, Switzerland). Several fractions were isolated and characterized by real-time PCR for the presence of limbal SC markers and differentiation markers of corneal epithelial cells by flow cytometry for the determination of the side-population (SP) phenotype and growth properties in vitro.

RESULTS. Cells retained in the lightest fraction (40% Percoll) and in the densest fraction (80% Percoll) of the gradient were both enriched for populations with a high expression of the SC markers ABCG2 and Lgr5 and also expressing the SP phenotype. However, the lightest fraction (representing approximately 12% of total limbal cells) contained cells with the strongest spontaneous proliferative capacity and expressed the corneal epithelial differentiation marker K12. In contrast the densest fraction (<7% of original cells) was K12 negative and contained small nonspontaneously proliferating cells, which instead were positive for p63. Unexpectedly, cells from this fraction had the highest proliferative activity when cultured on a 3T3 feeder cell monolayer.

CONCLUSIONS. These findings demonstrate the presence of two distinct populations of corneal epithelial cells with limbal SC characteristics, based on differential expression of the keratin-specific marker K12 and transcription factor p63, and suggest a difference in developmental stage of the two populations, with the K12–p63+ population being closer to the primitive limbal SC.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2008 by the Association for Research in Vision and Ophthalmology