Genetics of corneal endothelial dystrophies

The corneal endothelium maintains the level of hydration in the cornea. Dysfunction of the endothelium results in excess accumulation of water in the corneal stroma, leading to swelling of the stroma and loss of transparency. There are four different corneal endothelial dystrophies that are hereditary, progressive, non-inflammatory disorders involving dysfunction of the corneal endothelium. Each of the endothelial dystrophies is genetically heterogeneous with different modes of transmission and/or different genes involved in each subtype. Genes responsible for disease have been identified for only a subset of corneal endothelial dystrophies. Knowledge of genes involved and their function in the corneal endothelium can aid understanding the pathogenesis of the disorder as well as reveal pathways that are important for normal functioning of the endothelium.     

STUDIES ON THE CORNEA : I. The Fine Structure of the Rabbit Cornea and the Uptake and Transport of Colloidal Particles by the Cornea in Vivo

Physiological studies have demonstrated that ions, as well as large molecules such as hemoglobin or fluorescein, can diffuse across and within the cornea. Most of the substrates for corneal metabolism are obtained from aqueous humor filling the anterior chamber. In order to receive its nutrients and in order to maintain its normal conditions of hydration, the avascular cornea must transport relatively large amounts of solute and solvent across the cellular layers which cover this structure. It has been suggested in the past that there may be a morphological basis for the transport of large amounts of solvents and solutes by cells by the mechanism of pinocytosis. The use of electron-opaque markers to study fluid movements at the electron microscope magnification level was described by Wissig (29). The present study describes the fine structure of the normal rabbit cornea and the pathways of transport of colloidal particles by the cornea in vivo. Rabbit corneas were exposed in vivo to suspensions of saccharated iron oxide, thorium dioxide, or ferritin by injection of the material into the anterior chamber. In other experiments thorium dioxide or saccharated iron oxide was injected into the corneal stroma, producing a small bleb. Particles presented at the aqueous humor surface of the rabbit corneal endothelium are first attached to the cell surface and then pinocytosed. It appears that the particles are carried around the terminal bar by an intracellular pathway involving the pinocytosis of the particles and their subsequent transport in vesicles to the lateral cell margin basal to the terminal bar. Particles introduced at the basal surface of the endothelium (via blebs in the corneal stroma) are apparently carried through the endothelial cells in membrane-bounded vesicles without appearing in the intercellular space. There appears to be free diffusion of these particles through Descemet's membrane and the corneal stroma. The stromal cells take up large quantities of the particles when blebs are injected into the stroma.     

Clinical cryobiology of tissues: preservation of corneas

It is well recognized that the clarity of the cornea is a function of its hydration, and that this hydration is controlled by a "pump-and-leak" mechanism operating across the posterior monolayer of cells called the endothelium. A breakdown of the endothelium through disease or injury causes a marked increase in corneal thickness as the stroma imbibes fluid from the aqueous humor in the anterior chamber of the eye. This thickened, edematous condition of the stroma results in a cloudy cornea with an associated marked decrease in visual acuity. Treatment for this condition is usually by full-thickness corneal transplantation (penetrating keratoplasty), the success of which is dependent upon the donor cornea having an intact and healthy endothelium. It is essential, therefore, that any method of corneal storage for penetrating keratoplasty should protect and preserve the endothelium in a viable state. Current clinical practice relies upon short-term methods of preservation by two principal methods. Moist Chamber Storage is the time-honored corneal preservation method; it consists of keeping enucleated eyes at 0-4 degrees C in a sealed jar containing a pad of cotton gauze soaked in saline to provide a humid environment. The time limit placed upon this method of storage is 24-48 hr after which the viability of the endothelium deteriorates rapidly. Storage in M-K (McCarey-Kaufman) Medium involves excision of the corneoscleral segment from the donor eye and immersing it, endothelial side uppermost, in a medium consisting of tissue culture medium, 5% Dextran 40, and antibiotics. Laboratory and clinical studies indicate that storage in M-K medium at 4 degrees C preserves human endothelial cells for up to 4 days when the eye has been removed from the cadaver in less than 10 hr postmortem. Long-term preservation of corneas by freezing has long been a major goal in eye banking because indefinite storage by cryopreservation offers significant advantages for the quality and the quantity of material for use in keratoplasty, as well as for its distribution. However, procedures that have been developed for the cryopreservation of corneas have not been widely used, and a number of studies have shown that these procedures are inadequate for maintaining the integrity of the corneal endothelium. Not surprisingly, clinicians are now reluctant to accept corneas that have been frozen by these methods, though the clinical need is now greater than ever.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rabbit Corneal Hydration and the Bicarbonate Pump

Experiments were conducted on the transport properties of the rabbit corneal endothelium at 22 °C, at which temperature the endothelium was able to stabilize the hydration of corneal stroma at physiological values. When bicarbonate was omitted from the bathing solution, the cornea swelled at 11 ± 1 μm.h⁻¹. The swelling was completely reversible upon the subsequent re-introduction of bicarbonate. Similar swelling rates were observed when the endothelial pump was irreversibly inhibited with ouabain. In an Ussing-type chamber, the endothelium developed an electrical resistance of 25.0 ± 1.0 Ω.cm² and a short circuit current (s.c.c.) of 6.0 ± 1.1 μA.cm⁻². Neither electrical resistance of the corneal endothelium nor its s.c.c. were changed significantly after exposure to 0.5 mM amiloride. Ouabain abolished the s.c.c. but had no significant effect on resistance. When paired preparations were short-circuited, the endothelium developed a net H[¹⁴C]O ₃ ⁻ flux of 0.24 ± 0.03 μmoles.cm⁻².h⁻¹ into the aqueous humour, which was close in magnitude and direction to the s.c.c. of 0.22 ± 0.01 μEq.cm⁻².h⁻¹. There was no significant net flux of ⁸⁶Rb (0.04 ± 0.03 μmoles.cm⁻².h⁻¹). Similar magnitude fluxes for both bicarbonate and rubidium were found with open-circuit preparations. It is suggested that a metabolically driven electrogenic bicarbonate current passing across the corneal endothelium is solely responsible for maintaining corneal hydration at 22 °C. Based on these and other studies, a model is proposed for active bicarbonate transport across corneal endothelium consisting of uphill entry into the cell through a baso-lateral membrane sodium/bicarbonate cotransporter (NBC) and downhill exit through an apical membrane anion channel. Studies on the transport properties of the endothelium at 35 °C are discussed and reasons suggested for the discrepancy between short circuit current and net bicarbonate flux at this closed eye temperature.     

Aquaporin deletion in mice reduces corneal water permeability and delays restoration of transparency after swelling

Two aquaporin (AQP)-type water channels are expressed in mammalian cornea, AQP1 in endothelial cells and AQP5 in epithelial cells. To test whether these aquaporins are involved in corneal fluid transport and transparency, we compared corneal thickness, water permeability, and response to experimental swelling in wild type mice and transgenic null mice lacking AQP1 and AQP5. Corneal thickness in fixed sections was remarkably reduced in AQP1 null mice and increased in AQP5 null mice. By z-scanning confocal microscopy, corneal thickness in vivo was (in microm, mean +/- S.E., n = 5 mice) 123 +/- 1 (wild type), 101 +/- 2 (AQP1 null), and 144 +/- 2 (AQP5 null). After exposure of the external corneal surface to hypotonic saline (100 mosm), the rate of corneal swelling (5.0 +/- 0.3 microm/min, wild type) was reduced by AQP5 deletion (2.7 +/- 0.1 microm/min). After exposure of the endothelial surface to hypotonic saline by anterior chamber perfusion, the rate of corneal swelling (7.1 +/- 1.0 microm/min, wild type) was reduced by AQP1 deletion (1.6 +/- 0.4 microm/min). Base-line corneal transparency was not impaired by AQP1 or AQP5 deletion. However, the recovery of corneal transparency and thickness after hypotonic swelling (10-min exposure of corneal surface to hypotonic saline) was remarkably delayed in AQP1 null mice with approximately 75% recovery at 7 min in wild type mice compared with 5% recovery in AQP1 null mice. Our data indicate that AQP1 and AQP5 provide the principal routes for corneal water transport across the endothelial and epithelial barriers, respectively. The impaired recovery of corneal transparency in AQP1 null mice provides evidence for the involvement of AQP1 in active extrusion of fluid from the corneal stroma across the corneal endothelium. The up-regulation of AQP1 expression and/or function in corneal endothelium may reduce corneal swelling and opacification following injury.     

STUDIES ON THE CORNEA : II. The Uptake and Transport of Colloidal Particles by the Living Rabbit Cornea in Vitro

In vitro studies of the transport of colloidal particles by the cornea were carried out on intact corneas of adult rabbits in a chamber described by Donn, Maurice, and Mills (2) in which the epithelial or the endothelial surface of the cornea was exposed to thorium dioxide or saccharated iron oxide under various conditions. These studies confirmed the results of previous work in vivo and allowed modification of the experimental conditions. Particles are pinocytosed at the apical surface of the corneal endothelium and carried around the terminal bar in membrane-bounded vesicles. Basal to the terminal bar these vesicles fuse with the lateral cell margin and their contents are released into the intercellular space, in which they appear to be carried by a one-way flow down to Descemet's membrane and the corneal stroma. Indications that the endothelial transport is an active process are presented by the different pathways of transport into or out of the corneal stroma, as well as by the approximately 70 per cent reduction in transport activity at low temperatures.     

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Cl- cotransport

The role ofNa⁺-K⁺-2Clcotransport in ion and fluid transport of the corneal endothelium wasexamined by measuring changes in corneal hydration and uptake of⁸⁶Rb by the endothelial celllayer. Isolated, intact rabbit corneas maintain normal hydration whenthey are superfused at the endothelial surface with bicarbonate()-Ringer solutions as aresult of equilibrium between active ion and fluid transport out of thestromal tissue and leak of fluid into stromal tissue from the aqueoushumor. Furosemide and bumetanide did not alter this equilibrium whenthey were added to the superfusion medium. Uptake of⁸⁶Rb by the endothelium of theincubated cornea was increased 25% by bumetanide, but uptake in thepresence of ouabain (70% less than that of controls) was not changedby bumetanide. In Na⁺-free medium,uptake of ⁸⁶Rb was reduced by58%, but it was unchanged inCl-free medium. CalyculinA, a protein phosphatase inhibitor and activator ofNa⁺-K⁺-Clcotransport, was without effect on⁸⁶Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes wassignificantly different when bumetanide was present in the media. It isconcluded thatNa⁺-K⁺-2Clcotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport ofthis cell layer. Its presence in cultured endothelial cells may reflectthe reported importance of this protein in growth, proliferation, anddifferentiation.

     

Role of computer-assisted analysis of the corneal endothelium in vitreoretinal surgery with intraocular silicone oil injection: a technical report

The innermost lining of the cornea consists of a single layer of cells called the endothelium. Despite its name, the endothelium of the cornea differs considerably from the vascular endothelium, both functionally and morphologically. The corneal endothelium plays a fundamental role in maintaining the transparency of the corneal membrane, as the result of both its function as a barrier against penetration of the aqueous humor in the parenchyma and its ability to remove water from the stroma (usually referred to as the endothelial "pump" function). Any abnormality in the corneal endothelium causes, first, the impairment of its function as a barrier and pump due to the loss of stromal anti-turgor mechanisms, followed by edema and possible development into keratopathy. The specular microscope is an instrument which makes it possible to see the endothelial "mosaic" in the reflected image of the posterior corneal surface. A large variety of clinical specular microscopes is presently available, both contact and non-contact, which allow, for easy and rapid photography of the corneal endothelium "in vivo". In the present case, we used a non-contact computerized specular microscope to analyze the corneal endothelium in a group of patients affected by retinal detachment who needed to undergo vitreoretinal surgery with immission of silicone oil into the vitreal chamber.     

The Balance of Fluid and Osmotic Pressures across Active Biological Membranes with Application to the Corneal Endothelium

The movement of fluid and solutes across biological membranes facilitates the transport of nutrients for living organisms and maintains the fluid and osmotic pressures in biological systems. Understanding the pressure balances across membranes is crucial for studying fluid and electrolyte homeostasis in living systems, and is an area of active research. In this study, a set of enhanced Kedem-Katchalsky (KK) equations is proposed to describe fluxes of water and solutes across biological membranes, and is applied to analyze the relationship between fluid and osmotic pressures, accounting for active transport mechanisms that propel substances against their concentration gradients and for fixed charges that alter ionic distributions in separated environments. The equilibrium analysis demonstrates that the proposed theory recovers the Donnan osmotic pressure and can predict the correct fluid pressure difference across membranes, a result which cannot be achieved by existing KK theories due to the neglect of fixed charges. The steady-state analysis on active membranes suggests a new pressure mechanism which balances the fluid pressure together with the osmotic pressure. The source of this pressure arises from active ionic fluxes and from interactions between solvent and solutes in membrane transport. We apply the proposed theory to study the transendothelial fluid pressure in the in vivo cornea, which is a crucial factor maintaining the hydration and transparency of the tissue. The results show the importance of the proposed pressure mechanism in mediating stromal fluid pressure and provide a new interpretation of the pressure modulation mechanism in the in vivo cornea.     

Application of catastrophe theory to corneal swelling

Stromal swelling in human, cat, and rabbit cornea is biphasic, interpretable as an elementary cusp catastrophe proposed by Thom, with t* = log t and Q* = log Q (stromal charge Q, time t) as control parameters, and H0.5 (hydration H) as the state variable. A thermodynamic potential with two attractor regions, each with a local minimum, governs corneal stromal swelling. Transitions follow a 'saturation convention' whereby the second minimum is preferred upon availability. Corneal swelling is an example of a space-equivalent unfolding, where the transition plane moves in time. It is proposed that the transition plane coincides with the uncoupling of interfibrillary linkages or 'springs' in the corneal stroma, and is associated with a critical hydration of ca. 10 kg H2O per kilogram dry mass, and stromal charge ca. 1 x 10(-7) mol electrons.     

Corneal endothelial structure and function under normal and toxic conditions.

Our understanding of the function of the corneal endothelium in corneal thickness regulation, and the role of ion transport mechanisms in endothelial physiology, has expanded greatly over the past 25 years. The basic events occurring across the apical and basolateral membranes of the cells are far better understood today, although gaps still exist in the area of the relationship of the cellular and paracellular pathways and their relative contribution to the overall behavior of the endothelium. Little is known about the movement of ions or fluid between the cells or in what proportion this may occur compared to the cellular events. Furthermore, although our knowledge of the ionic movement processes has been enhanced, the link between fluid transfer across the endothelium and ion movements remains an enigma. Important questions also remain concerning the link between electrical characteristics and either ion movement or fluid transport. Improved storage solutions are needed that will preserve endothelial function after transplantation through the provision of a significant improvement in long-term cell survival. The limit to preservation time at present is about 14 days, and the use of other variables in the storage solution may extend this time. In reality, however, extension of preservation time is now of secondary importance relative to the need to enhance cell survival and reduce cell loss following surgery. Whether such improvement can be made with manipulation of the solution alone, or whether refinements are needed in the surgical technique awaits further study. Our comprehension of the biochemical linkage between energy supply and ion movement also remains uncertain in view of the particular intracellular localization of the anionic ATPases to mitochondrial loci. Despite numerous attempts there have been only a few chemicals identified that stimulate the fluid pump, but the level of stimulation has been relatively small and short-lived. No sustained effects have been found that would be of clinical benefit in reducing corneal thickness. A considerable variety of chemicals has been tested on the endothelium and it is unlikely that any new compounds will be identified that will cause enhancement of the fluid pump that would be of clinical benefit in dystrophic, or otherwise swollen, corneas. Of all the toxic responses of the endothelium the majority have been identified because of a malfunction of corneal thickness regulation, with the resultant corneal swelling, or by morphological examination. Only in a few instances has the permeability to non-electrolytes (carboxyfluorescein, inulin/dextran) been measured, and even more rarely have ion fluxes, or pump activity (3H-ouabain binding), been measured.(ABSTRACT TRUNCATED AT 400 WORDS)     

Propane-1,2-diol as a potential component of a vitrification solution for corneas

Any method of cryopreservation of the cornea must maintain integrity of the corneal endothelium, a monolayer of cells on the inner surface of the cornea that controls corneal hydration and keeps the cornea thin and transparent. During freezing, the formation of ice damages the endothelium, and vitrification has been suggested as a means of achieving ice-free cryopreservation of the cornea. To achieve vitrification at practicable cooling rates, tissues must be equilibrated with high concentrations of cryoprotectants. In this study, the effects of propane-1,2-diol on the structure and function of rabbit corneal endothelium were studied. Corneas were exposed to concentrations of propane-1,2-diol ranging from 10 to 30% v/v in a Hepes-buffered Ringer's solution containing glutathione, adenosine, 5 mmol/liter sodium bicarbonate, and 6% w/v bovine serum albumin. Endothelial function was assessed by monitoring corneal thickness during perfusion of the endothelial surface at 34 degrees C for 6 hr. Exposure to 10-15% v/v propane-1,2-diol was well tolerated for 20 min at 4 degrees C when the cryoprotectant was removed in steps or by sucrose dilution. However, exposure to 25% v/v propane-1,2-diol for 20 min at 0 or -5 degrees C was consistently tolerated only when 2.5% w/v chondroitin sulfate was included in the vehicle solution. Exposure to 30% v/v propane-1,2-diol was harmful at -5 and -10 degrees C. The endothelial damage following exposure to 30% v/v propane-1,2-diol was probably the result of a toxic effect rather than osmotic stress. Although 25% v/v propane-1,2-diol does not vitrify at cooling rates that are practicable for corneas, it could at this concentration form a major component of a vitrification solution comprising a mixture of cryoprotectants.     

Swelling studies on the cornea and sclera: the effects of pH and ionic strength.

The biophysical properties of the cornea and sclera depend on the precise maintenance of tissue hydration. We have studied the swelling of the tissues as a function of pH and ionic strength of the bathing medium, using an equilibration technique that prevents the loss of proteoglycans during swelling. Synchrotron x-ray diffraction was used to measure the average intermolecular and interfibrillar spacings, the fibril diameters, and the collagen D-periodicity. We found that both tissues swelled least near pH 4, that higher hydrations were achieved at lower ionic strengths, and that sclera swelled about one-third as much as cornea under most conditions. In the corneal stroma, the interfibrillar spacing increased most with hydration at pH values near 7. Fibril diameters and D-periodicity were independent of tissue hydration and pH at hydrations above 1. Intermolecular spacings in both tissues decreased as the ionic strength was increased, and there was a significant difference between cornea and sclera. Finally, we observed that corneas swollen near pH 7 transmitted significantly more light than those swollen at lower pH levels. The results indicate that the isoelectric points of both tissues are close to pH 4. The effects of ionic strength can be explained in terms of chloride binding within the tissues. The higher light transmission achieved in corneas swollen at neutral pH may be related to the fact that the interfibrillar fluid is more evenly distributed under these conditions.     

STUDIES ON THE CORNEA : III. The Fine Structure of the Frog Cornea and the Uptake and Transport of Colloidal Particles by the Cornea in vivo

The fine structure of the frog cornea has been studied and compared with that of the rabbit cornea (1, 2) particularly in relation to the uptake and transport of colloidal particles. The frog corneal endothelium does not possess a terminal bar and the fluid space of the intercellular space is apparently continuous with that of the anterior chamber. Colloidal markers (ThO₂, Fe₂O₃) placed in the anterior chamber pass down the intercellular space into the cornea. Markers injected intrastromally diffuse freely in the stroma and Descemet's membrane but pass across the endothelium only via membrane-bounded vesicles. These results are compared with those of similar experiments in the rabbit and it is concluded that the primary pathway for the passage of materials into the cornea is intercellular and that the pinocytotic pathway of the rabbit corneal endothelium (Kaye and Pappas; Kaye et al.) is an adaptation to the presence of a terminal bar. The significance of the separation of inward and outward pathways in terms of corneal metabolism is considered.     

Aquaporins and CFTR in Ocular Epithelial Fluid Transport

Aquaporins (AQPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) provide the molecular routes for transport of water and chloride, respectively, through many epithelial tissues. In ocular epithelia, fluid transport generally involves secondary active chloride transport, which creates the osmotic gradient to drive transepithelial water transport. This review is focused on the role of AQPs and CFTR in water and ion transport across corneal/conjunctival epithelia, corneal endothelium, ciliary epithelium, and retinal pigment epithelium. The potential relevance of water and chloride transport to common disorders of ocular fluid balance is also considered. Recent data suggest AQPs and CFTR as attractive targets for drug development for therapy of keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment, and retinal ischemia.     

Recovery of Corneal Endothelial Cells from Periphery after Injury

Background

Wound healing of the endothelium occurs through cell enlargement and migration. However, the peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium in endothelial injury.

Aim

To investigate the recovery process of corneal endothelial cells (CECs) from corneal endothelial injury.

Methods

Three patients with unilateral chemical eye injuries, and 15 rabbit eyes with corneal endothelial chemical injuries were studied. Slit lamp examination, specular microscopy, and ultrasound pachymetry were performed immediately after chemical injury and 1, 3, 6, and 9 months later. The anterior chambers of eyes from New Zealand white rabbits were injected with 0.1 mL of 0.05 N NaOH for 10 min (NaOH group). Corneal edema was evaluated at day 1, 7, and 14. Vital staining was performed using alizarin red and trypan blue.

Results

Specular microscopy did not reveal any corneal endothelial cells immediately after injury. Corneal edema subsided from the periphery to the center, CEC density increased, and central corneal thickness decreased over time. In the animal study, corneal edema was greater in the NaOH group compared to the control at both day 1 and day 7. At day 1, no CECs were detected at the center and periphery of the corneas in the NaOH group. Two weeks after injury, small, hexagonal CECs were detected in peripheral cornea, while CECs in mid-periphery were large and non-hexagonal.

Conclusions

CECs migrated from the periphery to the center of the cornea after endothelial injury. The peripheral corneal endothelium may act as a cell resource for the recovery of corneal endothelium.     

Enhanced detection method for corneal protein identification using shotgun proteomics

Background  

The cornea is a specialized transparent connective tissue responsible for the majority of light refraction and image focus for the retina. There are three main layers of the cornea: the epithelium that is exposed and acts as a protective barrier for the eye, the center stroma consisting of parallel collagen fibrils that refract light, and the endothelium that is responsible for hydration of the cornea from the aqueous humor. Normal cornea is an immunologically privileged tissue devoid of blood vessels, but injury can produce a loss of these conditions causing invasion of other processes that degrade the homeostatic properties resulting in a decrease in the amount of light refracted onto the retina. Determining a measure and drift of phenotypic cornea state from normal to an injured or diseased state requires knowledge of the existing protein signature within the tissue. In the study of corneal proteins, proteomics procedures have typically involved the pulverization of the entire cornea prior to analysis. Separation of the epithelium and endothelium from the core stroma and performing separate shotgun proteomics using liquid chromatography/mass spectrometry results in identification of many more proteins than previously employed methods using complete pulverized cornea.     

Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells

In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."