IN-VITRO PROPAGATION OF HUMAN OCULAR SURFACE EPITHELIAL-CELLS FOR TRANSPLANTATION

Purpose. To examine the possibility that ocular surface epithelial cel ls might be grown in culture for use as grafts. Methods. The prolifera tive capacity of epithelial cells cultured from the conjunctiva, limbu s, and central cornea of normal human eyes was compared. Single cells disaggregated from approximately 1 mm2 biopsy specimens were serially cocultured with lethally irradiated mouse 3T3 fibroblasts. To study th e cells' ability to reform a stratified epithelium, confluent limbal c ultures were released as an intact cell sheet with the enzyme. Dispase and transplanted to a dermal connective tissue bed in nude mice. Atta chment and differentiation properties of the reconstituted epithelium were examined immunohistochemically. Results. Central corneal epitheli al cells could not be propagated; they senesced in first or second pas sage. In contrast, limbal epithelial cells exhibited a substantial (i. e., mean of 23 population doublings) and conjunctival cells a moderate (i.e., mean of 11 population doublings) proliferative capacity. Withi n 4 days of transplantation to the nude mouse dermis, cultured limbal epithelial cells formed an epithelium 5-6 cell layers thick. The epith elium adhered firmly to the graft bed, and deposition of the basement membrane and anchoring fibril protein collagens IV and VII and laminin was detectable immunohistochemically. The transplanted epithelium dis played limbuslike compartmental expression of keratins K3, K13, and K1 9, and of the enzyme enolase. Conclusions. These results support the c oncept that corneal epithelial stem cells are located in the limbus an d indicate that cultured autologous limbal cells may function as graft s to permanently restore the corneal epithelium after severe ocular su rface injury.