title: A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient.
abstract: OBJECTIVE
  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.
authors.affiliation: Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Krulova
authors.forename: Katerina
authors.initials: K
authors.lastname: Pokorna
authors.forename: Anna
authors.initials: A
authors.lastname: Lencova
authors.forename: Jan
authors.initials: J
authors.lastname: Fric
authors.forename: Alena
authors.initials: A
authors.lastname: Zajicova
authors.forename: Martin
authors.initials: M
authors.lastname: Filipec
authors.forename: John V
authors.initials: JV
authors.lastname: Forrester
authors.forename: Vladimir
authors.initials: V
authors.lastname: Holan
mesh_terms: D016475:3T3 Cells; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics; D000818:Animals; D002455:Cell Division; D002469:Cell Separation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D019573:Epithelium, Corneal / Q000166:cytology*; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005434:Flow Cytometry; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D016133:Polymerase Chain Reaction; D011205:Povidone; D012822:Silicon Dioxide; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
chemical_list: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; C501412:Abcg2 protein, mouse; C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone