Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcription profiling by array of human conjunctival epithelial side population cells

ABSTRACT: Side population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival side population cells by global differential gene expression to identify markers and signaling pathways associated with this cell phenotype. Four overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 3342 were cytometrically sorted into SP and non-SP cohorts. RNA was isolated and processed for microarray analysis using a commercial oligonucleotide array representing more than 55,000 transcripts derived from about 30,000 different genes. Selected microarray results were validated at the gene and protein levels by quantitative PCR, and immunological methods. Data mining methods were used to identify cellular processes relevant for stem cell function. Comparative analysis of transcripts expression based on expression levels and present/absent software calls across 4 replicate experiments identified 16993 expressed conjunctival epithelial transcripts including 10,266 unique known genes. Of those genes, 1254 and 363 were over expressed (> 2-fold ) or under expressed (< 0.5-fold), respectively, in the SP. The overexpressed set included genes coding for non-epithelial genes (e.g., CD62E/E-selectin and CD93), genes that have been associated with stem cell function in other cellular systems, including several homeodomain genes and genes whose over- or under-expression may underpin the stem cell slow cycling phenotype ( e.g., dual specificity phosphatases and cyclin kinases). Experiment Overall Design: Whole human conjunctivae from unidentifiable cadaver donors, aged 55 to 65, were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) within 48 hours of collection. No donor details apart from age, sex, and cause of death were released. The use of human tissue in the study was in accordance with the provisions of the Declaration of Helsinki and sanctioned by the Institutional Review Board. Fresh rabbit tissue was obtained from local abattoirs within one hour of sacrifice. Unless stated otherwise all reagents were procured from Sigma (St. Louis, Mo). Experiment Overall Design: Eight microarray experiments using side population (Hoechst 33342-transporting cells; SP) or non side population (Hoechst 33342-stained cells; nSP) cells from four cadavers.

ORGANISM(S): Homo sapiens

SUBMITTER: J Mario Wolosin

PROVIDER: E-GEOD-12631 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Molecular profiling of conjunctival epithelial side-population stem cells: atypical cell surface markers and sources of a slow-cycling phenotype.

Akinci M A Murat MA Turner Helen H Taveras Maria M Barash Alex A Wang Zheng Z Reinach Peter P Wolosin J Mario JM

Investigative ophthalmology & visual science 20090325 9

<h4>Purpose</h4>Side-population (SP) cells isolated from limbal and conjunctival epithelia derive from cells that are slow cycling in vivo, a known feature of tissue stem cells. The purpose of this study was to define the molecular signature of the conjunctival SP cells and identify markers and signaling pathways associated with the phenotype of these cells.<h4>Methods</h4>Overnight cultures of freshly isolated human conjunctival epithelial cells stained with Hoechst 33342 were sorted by flow cy ...[more]

PMID: 19324848

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Project description:Tumours contain heterogeneous cell populations. A population enriched in tumour-initiating potential has been identified in soft-tissue sarcoma (STS) by the isolation of side population (SP) cells. In this study, we compared the gene expression profiles of SP and non-SP cells in STS and identified Hedgehog (Hh) and Notch pathways as potential candidates for the targeting of SP cells. Upon verification of the activation of these pathways in SP cells, using primary tumor xenografts in NOD-SCID mice as our experimental model, we used the Hh blocker Triparanol and the Notch blocker DAPT to demonstrate that the suppression of these pathways effectively depleted the abundance of SP cells, reduced tumour growth, and inhibited the tumour-initiating potential of the treated sarcoma cells upon secondary transplantation. The data provide additional evidence that SP cells act as tumour initiating cells and points to Hh and Notch pathways as enticing targets for developing potential cancer therapies. We used microarrays to detail the difference in gene expressions between the side population cells in soft-tissue sarcoma in comparison to the bulk non-side-population cells. To examine whether certain pathways may be differentially regulated in SP cells versus non-SP cells, which represent the bulk of tumour cells, we compared the gene expression profiles of SP and non-SP cells in four primary STS tumours each from different patient. Upon surgical excision, these tumours were dissociated mechanically and enzymatically into individual cells. Via Hoechst dye staining and flow-cytometry, these primary tumour cells were sorted into distinct side population and non-side population fractions. Total RNA is extracted from an equal number of SP and NSP cell from each primary tumour. cDNA from each sample was generated from the isolated total RNA and hybridized onto Affymetrix Human Genome EukGE-WS2v4 gene chips against the same reference cDNA library. After initial processing of the raw data, using the Genespring® GX software, the expression of SP cells from each tumour was normalized against the expression of the corresponding non-SP cells. A gene list was constructed by selecting genes that were regulated in the same direction (SP vs. NSP) in all sample pairs with a fold change greater than 1.25. This list was examined using Genespring® GX significant pathway function to identify differentially regulated pathways.

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Dataset Information

Transcription profiling by array of human conjunctival epithelial side population cells

Publications

Molecular profiling of conjunctival epithelial side-population stem cells: atypical cell surface markers and sources of a slow-cycling phenotype.

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