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L.K. Yeh, T. Kawakita, E.M. Espana, H. He, C.–Y. Liu, S.C. G. Tseng; Air Exposure Promotes Intrastromal Invasion of Rabbit limbal Epithelial Cells at the Limbal Location in Vivo . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1438.
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Purpose: Previously we have reported that in an organotypic culture rabbit corneal epithelial cells invade into the collagen gel, and such a phenomenon, termed intrastromal invasion, is markedly promoted by air–lifting and is fibroblast–dependent (Chen et al, EER1995;61:521–33). We speculate that intrastromal invasion may be a unique functional attribute of limbal stem cells and can be used as an assay to analyze limbal stromal niche. Methods:Each of 6 pigmented Dutch belt rabbit eyes was equally subdivided into 6 corneo–limbo–conjunctival explants, including 4 mm of the cornea and 4 mm of sclera with the pigmented limbus in the middle. These explants with or without prior digestion with dispase II were seeded on a polyester membrane (pore size is 3.0 µm) and cultured in submerged, air–lifted (moist), or air–lifted (dry) manner in SHEM for 7 days. In parallel, corneal or limbal epithelial sheets isolated by dispase digestion were recombined with live or dead limbal stroma, seeded on a polyester membrane, and cultured similarly in an air–dry manner. Numbers of epithelial intrastormal invasion were assessed by hematoxylin staining of tissue sections. Results: Intrastromal invasion of epithelial cells as islands or cords was noted at the limbal but not corneal region. Such invasion only occurred when undigested explants or tissue recombinants were cultured in an air–lifted (dry) manner, but not in a submerger manner. Furthermore, no intrastromal invasion was noted when the limbal epithelium was recombined with a dead limbal stroma. The intrastromal invasion was noted when limbal, but not corneal, epithelium, was recombined with the live limbal stroma. Conclusions: Collectively, the above findings support the concept that intrastromal invasion by epithelial cells happens in undigested tissue explants only when exposed to the air–fluid interface. This phenomenon exists uniquely for the limbal epithelium when in contact with the limbal stroma. Further characterization of the epithelial phenotype with respect to the expression of differentiation markers, cell adhesion molecules, and MMPs is underway and should shed lights to the mechanism underscoring how limbal epithelial stem cells are regulated by limbal stromal niche.
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