Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentiate into corneal epithelial-type cells

Purpose. Previous studies on the use of human amniotic membrane (HAM) in ra bbit stem cell deficiency models have found the new epithelium growing over the HAM to express cornea-specific keratins (K3 and K12) in 40% of the cas es, suggesting that HAM map have induced conjunctival epithelial cells to t ransdifferentiate into cornea-type epithelial cells. The current study was performed to determine whether HAM could induce transdifferentiation of con junctival epithelial cells when cultured in vitro. Methods, Conjunctival gr afts taken from the fornices of New Zealand white rabbits (6-12 weeks old) were placed over HAMs and lifted to an air-media interface using polypropyl ene double rings. These cultures were maintained in supplemented hormonal e pithelial medium with and without 3T3 feeder cells. Rabbit corneal epitheli al cells were cultured similarly using strips of keratolimbal grafts placed over HAM. The cultures were terminated at Various times between the 8th an d 15th day. The cultured epithelial cells were examined histologically and immunohistochemically using monoclonal antibodies AK-2 (to K12 keratin), AM -3 (to goblet cell mucin), and AE-5 (to K3 keratin). Results. Both conjunct ival and corneal epithelial cells cultured on HAMs showed multilayered, dif ferentiated epithelial structures. On immunohistochemical examination, both epithelial cells stained positive for AE-5. None of the cultured conjuncti val epithelial cells stained positively for AK-2, while the corneal epithel ial cells showed positive staining with AK-2. There were no AM-S-positive g oblet cells in either epithelial cell culture. There was no difference in t he immunohistochemical patterns between cultures with or without 3T3 feeder cells. However, culture without feeder cells seemed to manifest a more deg enerative appearance than those with feeders. Conclusion. HAM does not indu ce transdifferentiation of conjunctival epithelial cells into corneal-type epithelial cells under the in vitro culture conditions used in this study.