Abstract
Purpose: :
Although poorly defined, the niche is important for maintaining the stem cell function. To determine the role of fibroblasts in the limbal niche, we examined the modulating effect of amniotic membrane extract (AME) in clonal growth of limbal progenitors derived from limbal explant cultures.
Methods: :
Human limbal explants were cultured on plastic dishes in SHEM with or without 25 µg/ml AME. During the 14 days of cultivation, explant outgrowth was monitored for the presence of fibroblasts using phase-contrast microscopy and the remaining explant was removed for cross-sectioning and H&E staining. Outgrowing cells were assayed for formation of epithelial and fibroblast colonies with or without a 3T3 feeder layer. Their lysates were analyzed by Western blot for phosphorylation of three families of MAPK.
Results: :
In SHEM, the earliest outgrowth of limbal explants was seen in 2 explants on Day 3, and in all 6 explants on Day 4. However, in SHEM/AME, no outgrowth was seen on Day 3, but growth was noted in one explant on Day 4, and in all 6 explants on Day 6. By Day 14, all outgrowths in SHEM were larger than those in SHEM/AME as evidenced by a 3.6 fold increase of the total cell number. In SHEM, the outgrowing cells consisted of epithelial cells and a few fibroblast cells, which could form colonies after being passaged on plastic dishes without 3T3 feeder layers. In contrast, no fibroblasts were seen on the outgrowing cells cultured in SHEM/AME, and much fewer colonies of fibroblast cells were formed after being similarly passaged. Interestingly, less fibroblast remained in the limbal stroma of explants cultured in SHEM as compared to those cultured in SHEM/AME. The Western blot result showed that there was significant reduction of phosphorylation of p38 MAPK, but not JNK or ERK in lysates of outgrowing cells in SHEM/AME compared to those of SHEM. Furthermore, the total number of epithelial colonies derived from outgrowing cells per explant from SHEM/AME cultures were significantly more than that from SHEM/AME cultures (43.7±3.1 vs. 28.6±1.5, p=0.005).
Conclusions: :
By suppressing MAPK p38 phosphorylation, AM extract inhibited the migration of fibroblast cells from the limbal stroma which is potentially beneficial for maintaining the niche for limbal epithelial progenitor cells during ex vivo expansion from limbal explants.
Keywords: cornea: epithelium • cornea: stroma and keratocytes • proliferation