Immunohistochemical Localization and Expansion of Presumable Limbal Stem Cells From Feline Normal Cornea
N. Weichsler; V. Lavigne; L. Correa; H.D. Herrera
Limbal stem cell deficiency (LSCD) is a condition derived from total or partial destruction of the limbal cells which are responsible for renewing the corneal epithelium maintaining corneal transparency (Daniels et al 2006). LSCD is clinically associated to corneal opacity and vascularization due to corneal conjunctivalization resulting in visual impairment (Luengo Gimeno et al 2007). Alkali burns and feline herpes virus infection are conditions which can produce LSCD (Luengo Gimeno et al 2007, Sandmeyer et al 2005). Usually, the clinical management of LSCD is complicated and the final outcome is often unsuccessful despite of conventional treatments. Limbal transplantation should be an effective approach for resurfacing the cornea with corneal epithelium in cases of LSCD. Different techniques for limbal transplantation include grafts with conjunctival or corneal tissue as carrier, and transplantation of expanded stem-cells which requires the identification and culture of such a cells (Tseng & Tsai 1991, Akpek & Foster 1999). The purpose of this paper was to localize the presumable limbal stem cells in the feline cornea in order to use them for corneal transplantation in cases of LSCD.
Materials and Methods
Samples were obtained from 4 cadaverous donors cats using a 2mm dermatologic punch. The samples were taken from the superior limbus and central cornea in each case. The samples of 2 cats were process for histologic, immunohistochemical studies, and the other 2 for cell culture. For histological identification the samples were stain with hematoxylin and eosin (H/E) and Schiff periodic acid (PAS). For immunohistochemical studies the samples were processed in standard way and marked with antibodies anti pancreatin AE1-3, CK 5, CK 3/12. For culture, the samples of central cornea and limbus were seeded on a 3T3 irradiated feeder layer (Day 0). The cells were counted at day 7 and 20. After 3 days of culture a plate of 9,6 cm2 (5000 cells/cm2) was taken and seeded again.
Histological observation showed that the limbus presents a stratified epithelium with cuboid polygonal basal cells. This cells have similar morphology and localization than those identified as progenitor limbal cells in human corneoscleral limbus. The cell markers AE 1/3 and CK 5 were positive in central cornea and limbus. The central cornea expressed CK 3/12. Regarding cell expansion of limbus and corneal culture, in one of the cats we only first observed differences in growing obtaining a count of 580,000 cells at day 7 for limbus and no significant growth for central cornea. At day 20 the central cornea culture showed only 275,000 cells. In the second cat, a plate of 9.6 cm2 (5,000 cells/cm2) was taken from the culture of central cornea and limbus at the day 3 and seeded again. This procedure was made in order to have the same number of cell for each culture (N0) and be able to compare them. This was because, at the first seed (before expansion) it is impossible to know the number of cells that we have initially. Fourteen days after the culture (N14) the limbus showed 270,750 cells and central cornea 95,000 cells. Inside the different colonies the cells of the limbus were homogeneous, round shaped, and uniform in size. The cells of central cornea showed different sizes and shapes.
Discussion and Conclusions
The ocular surface referrers to the entire epithelial surface of the external eye, including the corneal epithelium and the bulbar and palpebral conjunctival epithelium. These two types of epithelium are arisen from two genotypically different cell types. The two most important differences between these types of epithelium are the presence of goblet cells in the conjunctival epithelium and the vascularization of the conjunctival stroma where this epithelium lies. The corneal epithelium is a transparent stratified squamous epithelium, lacking in goblet cells, with a cuboid basal layer lied on a non vascular stroma. The narrow transitional zone between the cornea and the conjunctiva is known as the limbus which is composed of several layers of epithelial cells and melanocytes. The limbal epithelium is considered to be the progenitor of the corneal epithelium but not of the conjunctival epithelium. The corneal epithelium is derived from limbal epithelial cells which are capable of "unlimited" self-renewal and upon division; these limbal cells, so-called stem cells, differentiate into transient amplifying cells (TAC) that have limited renewal capability, and proliferate into corneal epithelial cells. There is no report of limbal stem cells markers in dogs or cats. Because of this we considered the same cell markers reported for humans (Pellegrini et al 1999, Liu et al 2006, Vascotto et al 2006). The cell markers AE 1/3 and CK 5 are not specific markers for stem cells, but there were used as primary ones. CK 3/12 is a specific maker for corneal epithelium in humans. In humans, the superior limbus shows the major amount of stem cells (Pellegrini et al 1999) and this was the reason because we used the analogous area from limbal feline eye compared to the central cornea, which should present only differentiated epithelial corneal cells. These results support the idea that the feline limbus has a similar function than human limbus as progenitor of corneal epithelium. This was primarily demonstrated with results obtained by immunohistochemistry and culture.
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