A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
18469183
DOI
10.1167/iovs.08-1987
PII: iovs.08-1987
Knihovny.cz E-zdroje
- MeSH
- ABC transportér z rodiny G, člen 2 MeSH
- ABC transportéry analýza genetika MeSH
- buněčné dělení MeSH
- buňky 3T3 MeSH
- centrifugace - gradient hustoty metody MeSH
- fibroblasty cytologie MeSH
- kmenové buňky cytologie fyziologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- oxid křemičitý MeSH
- polymerázová řetězová reakce MeSH
- povidon MeSH
- průtoková cytometrie MeSH
- rohovkový epitel cytologie MeSH
- separace buněk metody MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ABC transportér z rodiny G, člen 2 MeSH
- ABC transportéry MeSH
- Abcg2 protein, mouse MeSH Prohlížeč
- oxid křemičitý MeSH
- Percoll MeSH Prohlížeč
- povidon 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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