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J. Li, J.B. Shen, R.W. Beuerman, D. Tan; Differential Expression of Putative Stem Cell Markers in Limbal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5155.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To differentiate quantitatively the association of gene expression of some putative stem cells markers with specific cell layers of the adult limbal epithelium. Methods: In situ isolation of limbal basal epithelial cells and upper layer cells by laser aided cell capture under high power microscope (PALM, Positioning and Ablation with Laser Microbeam) from OCT embedded human limbal rim (5 donors, 48 –72 hours post–mortal). Total RNA was extracted from the catapulted cells by Trizol reagent and subsequently RT and real time PCR analysis was carried out on cytokertin 3, connexin 43, ABCG2, p63 and Kit ligand. Results: Basal layer limbal epithelial cells (LB) and corresponding upper layer cells (LU) were precisely dissected from OCT embedded human limbal rims. On average, about 40ng of total RNA was obtained from 4000 cells pooled from several OCT sections. The precision of dissection was also confirmed by the low level expression of cytokeratin3 in LB (4.77%±0.039 of LU, n=4). The expression of connexin 43 was low in LB compared to LU as expected. The expression of ABCG2 and p63 was greater in LB with an average delta Ct (as compared to beta actin) of 7.44±0.7 (n=3) and 10.87±4.2 (n=5), while they were not detectable in LU. Kit ligand, a potential epithelial stem cell marker was found to be 1.93±0.72 (n=3) fold higher in the LB cells than limbal LU cells. Conclusions: Our study confirmed that the expression of p63, ABCG2 and Kit ligand is higher in the basal layer of limbal epithelium compared to more differentiated upper cell layers. Therefore, it is reasonable to use LB mRNA to obtain limbal stem cell markers although only part of these cells are believed to be true "stem" cells. However, the integrity of message has to be considered when searching for what are otherwise rare mRNAs.
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