A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
18469183
DOI
10.1167/iovs.08-1987
PII: iovs.08-1987
Knihovny.cz E-resources
- MeSH
- ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
- ATP-Binding Cassette Transporters analysis genetics MeSH
- Cell Division MeSH
- 3T3 Cells MeSH
- Centrifugation, Density Gradient methods MeSH
- Fibroblasts cytology MeSH
- Stem Cells cytology physiology MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Silicon Dioxide MeSH
- Polymerase Chain Reaction MeSH
- Povidone MeSH
- Flow Cytometry MeSH
- Epithelium, Corneal cytology MeSH
- Cell Separation methods MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- ATP Binding Cassette Transporter, Subfamily G, Member 2 MeSH
- ATP-Binding Cassette Transporters MeSH
- Abcg2 protein, mouse MeSH Browser
- Silicon Dioxide MeSH
- Percoll MeSH Browser
- Povidone MeSH
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.
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