STEM CELLS AND CORNEAL EPITHELIAL REGENERATION FRIEDRICH E. KRUSE Heidelberg, Germany SUMMARY movement of cells. The vertical movement can be docu­ Self-renewing tissues such as the corneal epithelium con­ mented experimentally by the chase of previously labelled tain stem cells which represent the proliferative reserve. basal cells and might be due to the proliferative pressure in Studies of cellular differentiation and proliferation sug­ the basal cell layer.5 The horizontal movement of corneal gest that corneal epithelial stem cells are localised exclus­ epithelial cells from the periphery to the centre was ively in the basal limbal epithelium. Although regulatory observed after experimental corneal epithelial wound­ factors for the amplification of corneal stem cells are ing.6.7 Similarly in rabbits, the centripetal replacement of unknown, serum factors such as retinoic acid might corneal epithelium after corneal grafts which originates in induce differentiation of stem cells to transient ampli­ the donor epithelium has been shown to start in the periph­ fying cells which are responsible for cell amplification. ery of the graft.R Tracing of peripherally located ink par­ These cells are regulated by various polypeptide growth ticles in the murine epithelium has proved that the factors and extracellular calcium. Loss or malfunction of centripetal movement of corneal epithelial cells also takes stem cells does not permit maintenance or regeneration of place under physiological circumstances in normal animal the corneal epithelial mass but leads to conjunctival­ corneas.9 Several observations indicate that the centripetal isation of the corneal surface. Clinically, several ocular movement exists in human corneas as well. First, corneal surface disorders such as chemical burns can cause lim­ erosions which do not include the limbal epithelium heal bal damage and consecutive limbal insufficiency. Treat­ in a centripetal fashion.lo Second, small subepithelial ment for these disorders is available only by cysts which develop between the sutures of corneal grafts transplantation of healthy stem cells, which can be per­ I I move towards the centre when the sutures are removed. formed as both autograft and allograft. Third, a centripetal movement of epithelial cells under physiological conditions was observed by specular 12 PRINCIPLES OF CORNEAL EPITHELIAL microscopy. REGENERATION The proliferation of basal cells as well as horizontal and Corneal epithelium is subject to a constant process of cell vertical cell movements have been summarised in kinetic renewal and regeneration. Cells in the uppermost layer of models which describe the maintenance of the corneal epi­ the corneal epithelium are continuously desquamated thelial mass. lUi While these mechanisms of the mainten­ from the surface and must be replaced by cell prolifer­ ance of corneal epithelium are generally accepted, the ation. The exclusive localisation of dividing cells in the question of the origin of corneal epithelial cell prolifer­ basal layer of the corneal epithelium suggests that pro­ ation has sparked a considerable controversy. Two oppos­ liferation is limited to basal cells. 1-4 Only cells which are ing theories exist, one of which claims that the origin of in contact with the basement membrane have the ability corneal epithelium is derived from the adj acent conjunc­ for mitotic cell division while cells which are displaced tiva by conjunctival transdifferentiation while the other into the suprabasal layers become post-mitotic and lose claims that the origin of corneal epithelial proliferation their capability for cell division.s depends on corneal stem cells in the limbal basal The kinetics of the maintenance of the corneal epithelial epithelium. mass is characterised by a vertical as well as a horizontal CONJUNCTIVAL Correspondence to; Dr med. Friedrich Eduard Kruse. Augenklinik TRANSDIFFERENTIATION der Universitat Heidelberg, 1m Neuenheimer Feld 400. 0-69120 Heidelberg, Germany. The first theory is based on early studies of corneal wound Eye (1994) 8,170-183 © 1994 Royal College of Ophthalmologists STEM CELLS OF THE CORNEAL EPITHELIUM 17 1 healing in humans which observed an ingrowth of con­ longer exposure to n-heptanol in combination with mech­ junctival epithelium onto the denuded cornea following anical scraping led to trans differentiation in only 32% of large epithelial wounds extending beyond the limbus.15.16 the eyes.20-27 In the remaining eyes conjunctival epithe­ Further observations and experimental studies showed lium did not transdifferentiate but was supported by neo­ that in the absence of vascular ingrowth conjunctival epi­ vascularisation. These findings suggest that the extent of thelium on the corneal surface loses its conjunctival phe­ removal of the corneal epithelium is responsible for the notype and becomes cornea-like.16-18 This transformation incidence of transdifferentiation or persistent conjunctiv­ of conjunctival epithelium to a corneal epithelium was alisation. This observation draws attention to the role of described by the term 'conjunctival transdifferentiation'. limbal epithelium in the maintenance and restoration of This phenomenon was investigated in numerous animal corneal epithelium. It is tempting to speculate that com­ studieslY-22 and led to the assumption that normal corneal plete removal of both corneal and limbal epithelium epithelium is maintained by the surrounding conjunctival induces irreversible conjunctivalisation. On the contrary, epithelium.13 incomplete removal of the limbal epithelium may allow However, two lines of evidence have cast doubt upon the reconstruction of the original epithelium aftera certain the existence of true conjunctival transdifferentiation. period which is needed for recovery of the damaged epi­ First, the histological and ultrastructural appearance of thelium and which can falsely be interpreted as the time transdifferentiated conjunctival epithelium differs from sequence of conjunctival transdifferentiation. that of genuine corneal epithelium, for example with To prove this hypothesis we investigated the interaction respect to the presence of goblet cells.23 Second, the phe­ between the duration of corneal exposure to n-heptanol notype of the epithelium after transdifferentiation shows and the extent of corneal and limbal epithelial removal by distinct differences from the phenotype of genuine corneal means of a histological survey.33 The results indicate that epithelium with respect to its metabolism as well as its exposure of the corneal epithelium to n-heptanol with composition of proteins and keratins.2�-26 These data indi­ mechanical scraping results in complete removal of the cate that conjunctival transdifferentiation does not repre­ epithelium when the agent is applied for more than 60 sent the true conversion of a differentiated conjunctival seconds. In contrast, the limbal epithelium was much phenotype into a differentiated corneal phenotype but more resistant to this treatment. Exposure of the limbal rather describes an environmental modulation of the con­ epithelium to n-heptanol resulted in incomplete removal junctival epithelium. This notion is supported by the of the basal layer even when the duration of treatment was observation that conjunctival transdifferentiation can be extended to 180 seconds. These results indicate that the inhibited by vascularisation.16.17 In contrast, occlusion of treatment used in the aforementioned investigations to the vessels in conjunctivalised epithelium induces con­ remove the corneal epithelium completely (n-heptanol junctival transdifferentiation.27 treatment of less than 120 seconds) most probably also These observations suggest that the blood vessels resulted in incomplete removal of the limbal basal supply conjunctival epithelium on the corneal surface epithelium. with substances which prevent transdifferentiation or To investigate further whether the remaining basal lim­ which are - in other words - important for the mainten­ bal epithelium retained its proliferative capacity despite ance of the conjunctival phenotype. Numerous studies by, the treatment we conducted tissue cultures of corneo­ for example, Tseng and coworkers have demonstrated that scleral specimens from eyes which had been treated with retinoic acid is of great importance for the differentiation n-heptanol for 60 and 90 seconds following mechanical of goblet cells under physiological conditions and that ret­ scraping of the epithelium." In these cultures we observed inoic acid can prevent conjunctival trans differentia­ a continuous outgrowth from the limbal epithelium onto tion.28-31 It is therefore tempting to speculate that a the denuded stroma. Immunohistochemical staining with localised deficiency of retinoic acid (and other unidenti­ various antibodies showed that the outgrowth was of lim­ fied factors) results in a loss of goblet cells and a modu­ bal derivation. These results indicate that the remaining lation of conjunctival epithelium into a cornea-like basal epithelium retains its proliferative capacity and epithelium. therefore can reconstitute an epithelial phenotype after A closer analysis of the experiments which were per­ removal of the corneal epithelium as observed in the afore­ formed to induce conjunctival transdif ferentiation suggest mentioned transdifferentiation experiments. In the light of another explanation for transdifferentiation in addition to these results experimentally induced trans differentiation environmental modulation.