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A. Deschambeault, P. Carrier, M. Talbot, L. Germain; In vitro Characterization of Human Limbal Epithelial Cells Isolated from the Four Quadrants . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1357.
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Purpose: The regeneration of the corneal epithelium rests on stem cells that are located in the limbus. The aim of the present study is to determine the proliferative potential as well as the ability to maintain an undifferentiated phenotype of human limbal epithelial cells (HLEC) in vitro after their isolation from the four quadrants of the eye. Methods: Corneas were cut into four quadrants to include the superior, temporal, inferior or nasal limbal zones. A 7.5 mm trepan was used to separate and remove the center of the cornea. HLEC, isolated from each quadrant of the tissue biopsies, were cultured in vitro. After each passage, the distribution of keratin 19 (labeling stem cells) and keratin 3 (differentiated cell marker) were determined for each quadrant by immunofluorescence. Cultured cells from each quadrant were also analyzed by flow cytometry (FACS) for the presence of K19, and Coulter counter to evaluate the size of the cells. The proliferative potential of each quadrant was measured. Cell morphology was also observed. Results: The proliferative potential of HLEC from the superior limbus (35 doubling populations) was higher than that of cells from the inferior, temporal and nasal limbus (25 doubling populations). Furthermore, in vitro, mean HLEC size increased following the passages from 13 µm (fresh cells) to 22 µm (senescent cells). HLEC from the superior limbus were smaller than cells from the other quadrants for high passages (P3-P6). Senescent cells were more abundant in the inferior, temporal and nasal quadrants than in the superior quadrants for high passages (P3-P6). By FACS analysis, the proportion of K19 expressing cells diminished with passages for all quadrants. By immunofluorescence studies, the expression of K3 increases with the passages for each quadrant. Conclusion: These results indicate that HLEC from the upper limbal corneal zone possess a higher proliferative potential, contain more cells with an undifferentiated phenotype, and suggest that the upper quadrant is the best site for harvesting biopsy to culture human epithelial cells for grafting.
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