Abstract
Purpose: :
Previously, we reported that air-lifting (AL), i.e., exposure to the air-medium interface, and intact amniotic membrane (iAM) are not prerequisites for intrastromal invasion by human limbal basal epithelial progenitor cells of cultured limbal explants. Because cross-section immunostaining showed that the invading progenitor cells expressed ΔNp63 (+)/pax6 (-) phenotype, we would like to further determine their clonal growth potential.
Methods: :
Human limbal explants were cultured on plastic (PL) dishes in SHEM with or without iAM, epithelially-denuded AM (dAM), or 25 µg/ml AM extract (AME). After 14 days of culture, the epithelial outgrowth and surface epithelium separated from PL and explant stroma by 10 mg/ml Dispase II overnight at 4° C were seeded at a density of 1 x 103 cells, while cells in the remaining stroma, released by 1 mg/ml Collagenase A overnight at 37° C were seeded completely on murine 3T3 feeder layers. The resultant epithelial clones were subjected to immunostaining using markers for epithelial progenitor and squamous metaplasia.
Results: :
On 3T3 feeder layers, the surface explant epithelium yielded 153, 74, 61, and 47 clones on PL, AME, dAM, and iAM, respectively, while the epithelial outgrowth yielded 44, 21, 18 and 16 clones on PL, AME, dAM, and iAM, respectively. Most clones were either Holoclones or Paraclones. In contrast, invaded epithelial cells only yielded 1-2 Meroclones with irregular borders and large cells. Furthermore, cells in these clones were positive for both ΔNp63 and pax6, indicating that they were derived from the ocular surface epithelial progenitor cells. Surprisingly, they were also positive for both K10 and K12, suggesting they underwent corneal epithelial differentiation with concomitant squamous metaplasia.
Conclusions: :
These results indicated that human limbal basal epithelial progenitor cells readily undergo intrastromal invasion at the limbus under different culturing conditions. Invaded epithelial progenitor cells also lose their clonal growth potential and undergo squamous metaplasia. Further study is underway to determine if such a phenomenon is further promoted by air-lifting, a condition recently reported leading to limbal epithelial squamous metaplasia.
Keywords: cornea: epithelium • cornea: stroma and keratocytes • cornea: basic science