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D.–Q. Li, Z. Chen, S.C. Pflugfelder; Characterization of Molecular Features and Pathway Profiles for Partially Enriched Putative Human Limbal Stem Cells by Affymetrix Microarray . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1204.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The concept that corneal epithelial stem cells reside in limbus has been recognized for more than a decade, but identification of these stem cells is challenging. This study characterized molecular features and pathway profiles of partially enriched putative human limbal stem cells by Affymetrix microarray. Methods: Primary cultured human limbal epithelial cells were selected into three populations by adhesion to collagen IV: 1) the rapidly adherent cells (RAC) within the first 20 minutes, 2) the slowly adherent cells (SAC) from 20 minutes to 2 hours, and 3) the non–adherent cells (NAC) after 2 hours. Five µg total RNA for each population was used for Affymetrix microarray with whole human genome GeneChip® U133 Plus 2.0. The array images were analyzed using Affymetrix GCOS software v1.2. The gene expression profiling was analyzed using the R package multcomp, GenMAPP 2.0 and MAPPFinder software. Results: Based on their phenotypes and growth potential, the RAC, SAC and NAC represent partially enriched putative stem cells, the transient amplifying cells (TAC) and terminally differentiated cells (TDC), respectively. The array data from duplicate experiments were highly reproducible (R2>0.95). Among 47,000 transcripts on a chip, about 19,500 transcripts (41.6±1.6%, n=6) were expressed by human limbal epithelial cells. 1958 transcripts, accounting for 10% of expressed genes, were significantly regulated among these 3 populations. When clustering the 292 genes filtered by 5–fold up– or down–regulation, the heatmap showed a unique expression pattern by RAC. There were 192 genes regulated in the same trend (46 up and 146 down) in RAC compared with both SAC and NAC. These genes include up–regulated growth arrest–specific 1, collagens, PDGF receptor and kinesin family, and down–regulated protein kinases, ubiquitin, SPRRs, interleukins and hypothetical proteins. Novel uses of gene ontology allowed functional validation and pathway profiling. In RAC, only 2 genes, CDKN1C and CDKN2B, were down– and 17 genes were up–regulated in cell cycle group; while 6 genes down– and 5 genes up–regulated in anti–apoptosis group, when compared with SAC and NAC. Conclusions: These findings reveal the molecular features and pathway profiles of the partially enriched putative human limbal stem cells, distinguishable from TAC and TDC. Further characterization of these exclusive regulated genes may facilitate identification of limbal stem cells.
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