Differential intrastromal invasion by normal ocular surface epithelia is mediated by different fibroblasts

For most mucosal epithelia including the ocular surface, it is generally believed that wound healing is executed by epithelial migration on the plane of erosion or ulceration. In explant cultures, we incidentally observed the phenomenon of intrastromal invasion by corneal, limbal and conjunctival epithelial cells even when cell migration on plastics was promoted. Homotypic and heterotypic tissue recombinants between corneal and conjunctival epithelial cells and their stroma revealed that this phenomena was dependent on viable mesenchymal cells and was more active in conjunctival stroma than corneal stroma. Using organotypic cultures in which 3T3 fibroblasts were incorporated in collagen gel, we noted that this phenomenon was fibroblast-dependent and up-regulated by lifting the culture to the air-fluid interphase. The extent of intrastromal invasion was decreased if 3T3 fibroblasts were treated with increasing concentrations of mitomycin C. The invading epithelial islands retained the same basal and suprabasal epithelial phenotypes as those of the surface epithelial layers using several anti-keratin monoclonal antibodies. Using 5-fluorouracil (5-FU) to eliminate the rapid-cycling, i.e. transient amplifying progenitor basal cells, we further noted that this phenomenon could still be produced by 5-FU-resistant slow-cycling progenitor cells of corneal, limbal and conjunctival explants. In organotypic cultures, conjunctival fibroblasts were more active than corneal fibroblasts in inducing corneal or conjunctival epithelial invasion. As such intrastromal invasion can experimentally be produced by normal non-transformed adult epithelial cells and mediated by fibroblasts, this in vitro phenomenon may be useful for studying the epithelial-mesenchymal interactions operating during embryonic development and post-natal wound healing.