Unknown,Transcriptomics,Genomics,Proteomics

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Comparative transcriptome profiling of human epidermis keratinocyte stem cells (KSC) versus keratinocyte progenitors (KP)

ABSTRACT: To characterize the global transcriptome of human keratinocyte stem cells (KSC) and keratinocyte progenitors (KP), primary basal keratinocyte subpopulations enriched in quiescent stem cells [Itg-?6bright / Trf-Rdim] or in cycling progenitors [Itg-?6bright / Trf-Rbright] were purified by flow cytometry from human skin samples. Sorted keratinocytes were then seeded in culture plates and maintained in culture medium overnight (15 hours), and genome-wide transcriptome analysis of these two subpopulations was directly performed, in order to globally maintain the initial phenotypes. After RNA preparation, gene profiling was performed using oligonucleotide microarrays (26068 probes). LOWESS normalisation was applied. Keywords: epidermis, keratinocyte stem cells, progenitors, transcriptome, stemness Dye-swap hybridization were performed. Slides were scanned with a Genepix 4000 microarray scanner (Axon Instruments, Molecular devices, Sunnyvale, CA). For each hybridized spot, the Cy3 and Cy5 fluorescence values were obtained by using Genepix Pro 4.0 software (Axon Instruments) and were saved as a result file

ORGANISM(S): Homo sapiens

SUBMITTER: Nicolas FORTUNEL

PROVIDER: E-GEOD-68583 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We used the MPSS technology to uncover gene expression profiling in a greater depth in the early embryonic gonads and primordial germ cells (PGCs) in the chicken. Total numbers of sequenced signatures were 1,012,533 and 995,676 for the PGCs and gonad. Using a false discovery rate cut-off of 0.05, we found 3.05 % of all signatures in the PCGs and 1.89 % of all signatures in the gonad signatures were significantly up-regulated compared to each sample. The MPSS result was very consistent with our previous result using EST data(http://chickgce.snu.ac.kr/). Keywords: cell type comparison Experimental animals provided for this experiment were maintained at the University Animal Farm, Seoul National University, and all experimental procedures were performed at the affiliated laboratories of the university. Gonadal cells were retrieved from the gonads of 6.5-day-old (stage 29) White Leghorn (WL) embryos by our standard procedure [26]. Embryos were freed from the yolk by rinsing with calcium- and magnesium-free PBS and the gonads were retrieved by dissection of embryo abdomen with sharp tweezers under a stereomicroscope. Embryonic gonads were collected from total 1,947 embryos by 10 highly-skilled persons through 8 separated experimental batches. Gonadal tissues were dissociated by gentle pipetting in 0.05% (v:v) trypsin solution supplemented with 0.53 mM EDTA. After centrifuged at 200xg for 5 min, total gonadal cells were loaded into MACS (Miltenyi Biotech, Germany), and the separated primordial germ cells (PGCs) were immediately stored in liquid nitrogen (-190ºC) until processed further. The numbers of PGCs in cell population before and after loading were counted. MACS treatment for chicken PGCs and counting PGC number Chicken gonadal cells were incubated with PGC-specific primary antibody, anti-stage specific embryo antigen (anti-SSEA)-1 antibody for chicken PGCs (mouse IgM isotype), for 20 min at the room temperature of 20-25?C. Anti-SSEA-1 antibody developed by [27] was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Development of Biological Science. After washing with 1mL buffer (PBS supplement with 0.5% BSA and 2mM EDTA), the supernatant was completely removed. The pellet was mixed with 100 ? buffer containing 20? of rat anti-mouse IgM microbeads for 15 min at 4?C. Treated cells were carefully washed by the addition of 500 ? buffer and subsequently loaded with MACS.[28] For counting cell number, chicken PGCs before or after MACS treatment were fixed with 1% (v:v) glutaraldehyde for 5 min and rinsed with 1x PBS twice. The anti-SSEA-1 ascites fluid diluted 1:1,000 in PBS was added and subsequent steps were carried out using DAKO universal LSAB® kit, Peroxidase (DAKO, USA) according to the manufacturer’s instruction. After 8 batches of cell preparation, total cell number of PGC-enriched fraction and gonadal stromal cells was 5.26X106 and 1.76X108, respectively. These cell populations were further used for total RNA isolation and massively parallel signature sequencing (MPSS) analysis.

Project description:Somatic mosaicism for DNA copy number alterations (SMC-CNA) is defined as gain or loss of chromosomal segments in mitotic cells within a single organism. As cells harboring SMC-CNA have the potential to undergo clonal expansion, SMC-CNA may be present in a substantial portion of cells in differentiated human tissues and may contribute to the predisposition of these cells to genetic disease including cancer. We characterized gross genomic alterations (>500 kbp) in uninvolved glandular tissue from 59 breast cancer patients and matched samples of primary tumors and lymph node metastases. Array based comparative genomic hybridization experiments showed 10% (6/59) of patients harbored 1 - 359 large SMC-CNA (mean: 1328 kbp; median: 961 kbp) in uninvolved glandular tissue. SMC-CNA were partially recurrent in tumors, albeit with considerable contribution of stochastic SMC-can, indicating genomic destabilization. Therefore, we hypothesized that the observed genomic destabilization is predetermined by mutations in genes related to maintenance of genomic integrity. Targeted resequencing of 301 known predisposition and somatic driver loci revealed mutations in the following genes: BRCA1 (p.Gln1756Profs*74, p.Arg504Cys), BRCA2 (p.Asn3124Ile), NCOR1 (p.Pro1570Glnfs*45), PALB2 (p.Ser500Pro) and TP53 (p.Arg306*). We demonstrated that gross SMC-CNA may be present in a substantial portion of glandular tissue cells, which are distant from that of the tumor cells, and may co-occur with point mutations in crucial cancer predisposing or somatic driver genes. Taken together, this highlights temporal and spatial neoplastic potential of uninvolved glandular tissue from breast cancer patients. Characterization of gross genomic alterations (>500 kbp) in uninvolved glandular tissue from 59 breast cancer patients and in the matched samples of primary tumors and lymph node metastases. Corresponding peripheral blood lekocyte DNA, a pool of female DNA (Promega), or uninvolved glandular tissue DNA was used as reference. No biological replicates are included.

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Comparative transcriptome profiling of human epidermis keratinocyte stem cells (KSC) versus keratinocyte progenitors (KP)

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