In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia: Implications on conjunctival epithelial transdifferentiation and stem cells

Purpose. The anterior surface of the eye is covered by several physically contiguous but histologically distinguishable epithelia overlying the cornea, limbus, bulbar conjunctiva, fornix conjunctiva, and palpebral conjunctiva. It is important to determine whether the different phenotypes of these epithelia are the result of intrinsic divergence, extrinsic modulation, or a combination of both. Based on keratin expression and cell kinetic criteria, the authors previously suggested that corneal epithelial stem cells may actually reside in the limbal basal layer. Methods. In this article, the relationship between the corneal-limbal epithelial cells and conjunctival epithelial cells was analyzed by comparing their growth and differentiation properties in an identical cell culture environment. Results. Using Dispase instead of trypsin to dissociate the cells, the authors were able to grow all five rabbit ocular surface epithelia in the presence of 3T3 feeder cells. They found that corneal and limbal cells synthesize identical keratins, including large amounts of the K3 and K12 markers of corneal-type differentiation. By contrast, all three conjunctival epithelia shared another keratin pattern, with large amounts of simple epithelial keratins but only minute amounts of K3/K12 keratins. Conclusions. This observation, coupled with previous findings that the 'transdifferentiation' of conjunctival epithelial cells to corneal epithelium appears to be both incomplete and reversible, provides strong evidence that (1) the limbal-corneal epithelial cells form a lineage distinct from the conjunctival lineage and (2) conjunctival transdifferentiation actually represents a process of environmental modulation. In addition, of the three types of conjunctival epithelial cells, fornix cells were found to have a much greater proliferative potential than bulbar and palpebral cells. This observation, coupled with recent finding that fornix is enriched in slow-cycling (label- retaining) cells, raises the possibility that conjunctival epithelial stem cells may preferentially reside in the fornix.