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M. Zhou, J. Xu, T.–T. Sun, R.M. Lavker; Gene Expression Profiles of Freshly Isolated Mouse Limbal and Corneal Epithelial Basal Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1205.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: It is well accepted that corneal epithelial stem cells are preferentially located in the basal layer of the limbal epithelium, whereas the corneal epithelial basal layer contains the transit amplifying cells (stem cell progeny). A lack of specific markers for limbal epithelial stem cells is a major impediment in their isolation and biochemical characterization. To identify molecular markers for limbal epithelial stem cells, we have analyzed the gene expression profiles of limbal and corneal epithelial basal cells that were freshly isolated from cryosections. Methods: Frozen sections (6 µm) of mouse limbal and corneal epithelia were subjected to laser capture microdissection (LCM) using a PALM MicroBeam system. As few as 50 limbal and corneal epithelial basal cells isolated from each mouse (n=6) yielded sufficient material for RNA extraction, 2 rounds of amplification, biotin labeling, and hybridization to Affymetrix cDNA GeneChips, which contained ∼30,000 mouse genes. Arrays were analyzed using Gene Ontology and dChip software to determine sample and gene clustering. The criteria used for gene selection was 2 fold or greater change and a p–value of 0.05, identifying ∼ 1000 expressed transcripts. Results: The LCM method that we employed obviated the need for tissue dehydration prior to microdissection. This enabled us to isolate quiescent limbal and corneal epithelial basal cells directly from frozen sections, thereby increasing the yield and quality of RNA. Sixty percent of the genes were up–regulated, and 40% were down–regulated in limbal basal comparing with corneal basal cells. Analysis of microarrays indicated that observed changes in transcript levels could be clustered into groups with specific functions (e.g., cell–cell adhesion, cell cycle regulation, signal transduction and transcription factors). Some of the up–regulated genes in limbal basal cells were expected (e.g., enolase), while others had not been previously characterized (e.g., transmembrane proteins, cell cycle inhibitors and anti–apoptosis genes). Conclusions: This LCM methodology allows us to obtain gene expression profiles from individual cells that are not affected by extrinsic manipulations (e.g., tissue processing, dissociation, culturing and/or cell sorting). Several of the genes that were preferentially expressed in limbal basal cells have also been reported in epidermal cell populations enriched in stem cells. This suggests the existence of a "signature" set of genes that may help define epithelial stem cells.
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