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A Deschambeault, PS Carrier, M Talbot, SL Guérin, FA Auger, L Germain; Regional Variation in the Localization of Limbal Stem Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1621.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Stem cells of the cornea are important for the regeneration of its epithelium. They are located in the limbus. The aim of the present study was to determine whether the limbal stem cells are distributed uniformly in the four quadrants of the eye. Methods: Corneas were cut into four quadrants that represent the superior, temporal, inferior and nasal limbus. We used a 7.5 mm trepan to separate and remove the center of the cornea. The origin of the Bowman's membrane was used to delineate the limbus from the cornea. Epithelial cells were isolated from each quadrant of the tissue biopsies, and either analyzed or cultured in vitro. The distribution of keratin 19 (stem cell marker) and keratin 3/12 (differentiated cell marker) were determined for each limbal quadrant by immunofluorescence and confocal microscopy. Fresh and cultured cells were analyzed by flow cytometry (FACS) for the presence of K19, and Coulter to evaluate the number and the shape of the cells. After each passage, cells were also analyzed by immunofluorescence (K19, K3/K12) and morphology. Results: FACS analyses revealed that a higher proportion of stem cells was found in the superior (51.7%±12.3) and in the inferior limbus (34.0%±14.8) than in the temporal (26.8%±11.8) and nasal (27.3%±11.2) limbus in K19 labeling of fresh cells. In addition, the Coulter Counter results revealed two distinct populations of cells with different diameter (8-10 mm and 14-16 mm) in variable proportions in the four quadrants. Moreover, cells isolated from the superior and inferior limbus were smaller and underwent more passages in culture. The proportion of K19 expressing cells diminished with passages. Furthermore, in situ, the respective patterns of expression of K19 and K3/K12 varied depending on the quadrant: a higher number of cells expressing K19 were present in the superior and inferior limbus compared to temporal and nasal limbus. Work is in progress to further study the expression patterns of K19 and K3/K12 by confocal microscopy analysis. Conclusion: Our results indicate that limbal stem cells are more abundant in the upper part of the cornea followed by the lower part. The fine localization of stem cells will be of practical importance for future clinical applications such as tissue harvesting for autologous stem cells transplantation or epithelial cell culture for grafting.
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