Abstract
Purpose: :
To investigate possible mechanism involved in the de-differentiation of human corneal epithelial cells in a defined low calcium and serum free medium
Methods: :
Human corneal epithelial cells were cultivated from fresh limbal tissue explants in two conditions: high calcium serum-containing SHEM and a defined, low calcium and serum free medium (CnT-20). After 2 weeks, they were passaged and seeded at two densities, low and high, in CnT-20 medium. Colony forming efficiency without any feeder layer was quantified in low density condition. Subsequent passages were performed for cells seeded at high density. Gene expression was analyzed by reverse transcription and real time PCR with TaqMan primers and probes. Protein localization was determined by immunofluorescent staining on corneoscleral tissue cryosections and in cell cultures.
Results: :
Corneal epithelial cells harvested from explant cultures in the low calcium serum-free defined medium CnT-20 generated 2 fold colony forming efficiency compared with those cultured in SHEM. Cells cultured in CnT-20 archived six passages while cells cultured in SHEM did not reach a third passage. Real time PCR analysis revealed that the cells cultured in the defined media expressed a higher mRNA levels of stem cell associated genes ABCG2 and p63 and lower levels of differentiation genes K12 and SPRR2B than cells grown in SHEM media, suggesting that this defined medium preserves more stem cell like characteristics than cells cultured in SHEM. When cells originally grown in SHEM were sub-cultured to CnT-20, stem cell associated genes expression levels were up-regulated and the cells could be continually passaged 4 times in CnT-20 medium. When those cells were returned to SHEM, differentiation occurred and the cells did not reach two passages. Further study demonstrated concomitant up-regulation of TCF4, DLL-1, GAS-1, p57 and NGF genes expression levels in more stem-like corneal epithelial progenitor cells. Immunofluorescent staining on corneal cryosections showed that TCF4, DLL-1 and NGF are exclusively expressed in the basal layer of the limbal epithelium.
Conclusions: :
These findings demonstrated that human corneal epithelial cells cultured in the defined media present stem-like progenitor cell characteristics. De-differentiation occurred when differentiated cells sub-cultured from SHEM to CnT-20 medium. The higher expression of TCF4, DLL-1, GAS-1, p57 and NGF genes in cells cultured in CnT-20 suggests a possible role in the mechanism for de-differentiation of human corneal epithelial cells.
Keywords: cornea: epithelium • differentiation • cornea: basic science