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S. Khashabi, Y. Liu, P. Nguyen, S. Yiu; Corneal Epithelial Cell and 3T3 Fibroblast Interactions: Evidence for Stem Cell Niche. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3756.
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Identify the presence of corneal epithelial cell and 3T3 fibroblast cell interactions as evidence of a limbal stem cell niche.
Mouse 3T3 fibroblasts were immunostained with a panel of antibodies before use as a feeder layer for corneal epithelial cells. 3T3 cells were treated with mitomycin C (MCC) and co-cultured with corneal epithelial cells. Chromosome analysis allowed identification of mouse 3T3 cells from human corneal cells. Immunostaining of 3T3 cells with the same antibody panel after exposure to epithelial cells was done.
The original immunofluorescent profile of the mouse 3T3 cells was negative for cytokeratin 3/12, connexin 43 and ABCG2 and positive for vimentin. The epithelial cells were co-cultured with MCC-treated 3T3 cells for 2 weeks. Chromosome analysis was used to identify which of the cultured cells were fibroblasts, and immunofluorescent staining was repeated. Staining revealed the 3T3 cells were now positive for cytokeratan 3/12, ABCG2, and connexin 43. Vimentin remained positive.
The immunofluorescent profile of the 3T3 fibroblast cells was altered after exposure to corneal epithelial cells. Cytokeratan 3/12 is a marker for differentiated epithelial cells. Fibroblast cells should be negative for such an epithelial-specific marker; however, the epithelial cells appear to have influenced/induced expression of this marker. ABCG2 and connexin 43 are positive in cells with proliferative potential. The fibroblasts originally had no proliferative potential, as was expected with MCC treatment; however, after 2 weeks of contact with the epithelial cells, the cells appeared to have reactivated and begun expressing these proliferative markers. Vimentin, a fibroblast marker, remained positive, verifying that the cells were fibroblasts. We suspect that the corneal epithelium, consisting of differentiated epithelial cells, transient amplified cells, and limbal stem cells creates a stem cell niche with the cells it comes into contact with, such as fibroblast cells, a component of corneal stroma.
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