A rapid separation of two distinct populations of mouse corneal epithelial cells with limbal stem cell characteristics by centrifugation on percoll gradient.

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.