Investigation Title Transcription profiling of hematopoietic and adipose tissue-derived stem cells Comment[Submitted Name] Hematopoietic vs adipose tissue-derived stem cells Experimental Design cell_type_comparison_design co-expression_design transcription profiling by array Experimental Design Term Source REF mo mo EFO Comment[ArrayExpressReleaseDate] 2006-10-15 Comment[AEMIAMESCORE] 5 Comment[ArrayExpressAccession] E-MEXP-872 Comment[MAGETAB TimeStamp_Version] 2010-08-11 16:21:51 Last Changed Rev: 13058 Experimental Factor Name cell type Experimental Factor Type cell_type Experimental Factor Term Source REF Person Last Name Shahdadfar Fronsdal Brinchmann Collas Tunheim Boquest Sigurjonsson Person First Name Aboulghassem Katrine Jan Philippe Siv Andrew Olafur Person Mid Initials B Person Email katrine.fronsdal@labmed.uio.no Person Phone +47 23071373 Person Fax + 47 23073510 Person Address Sognsvannvn. 20 Person Affiliation The National Hospital University of Oslo The National Hospital University of Oslo The National Hospital University of Oslo Institute of Basic Medical Sciences, University of Oslo, Norway SYM, Rikshospitalet, Oslo, Norway Institute of Basic Medical Sciences, University of Oslo, Norway The National Hospital University of Oslo Person Roles biomaterial_provider biomaterial_provider;submitter biomaterial_provider Person Roles Term Source REF mo mo;The MGED Ontology mo Quality Control Type biological_replicate Quality Control Term Source REF The MGED Ontology Replicate Type Replicate Term Source REF Normalization Type Normalization Term Source REF Date of Experiment Public Release Date 2006-10-15 PubMed ID Publication DOI Publication Author List Katrine Fronsdal; Aboulghassem Shahdadfar; Olafur Sigurjonsson; Andrew Boquest; Siv Tunheim; Philippe Collas; Jan Brinchmann Publication Title Publication Status Publication Status Term Source REF Experiment Description Comparison of the gene expression of the two different stem cell types: hematopoietic- and adipose tissue-derived stem cells. Protocol Name P-MEXP-32988 P-MEXP-32987 P-MEXP-5116 P-MEXP-5118 P-MEXP-5112 Affymetrix:Protocol:Hybridization-EukGE-WS2v4 P-AFFY-6 Affymetrix:Protocol:ExpressionStat Protocol Type grow specified_biomaterial_action hybridization feature_extraction bioassay_data_transformation Protocol Description Just after isolation these cells were frozen in liquid nitrogen. Then RNA was isolated for microarray. Treatment of hematopoietic stem cells Isolation of CD34+ Cells. Bone marrow samples were obtained by aspiration from consenting healthy adults. Mononuclear cells were isolated by density gradient centrifugation (1.077 g/ml) by using Lymphoprep (Nycomed, Oslo, Norway)and resuspended in RPMI medium 1640 (GIBCO,Carlsbad, CA, USA). CD34+ cells were isolated from these by using a commercial magnetic isolation technique (Direct CD34 Progenitor Cell Isolation Kit, Miltenyi Biotec,Bergisch Gladbach, Germany) according to the manufacturer's description. The isolated human CD34+ HSCs were labeled with anti-CD34 antibody(HPCA-2-PE, Becton Dickinson) and further purified by flow cytometry-activated cell sorting by using a Becton Dickinson FACSVantage cell sorter equipped with an argon ion laser. The final purity of the CD34+ population was more then 97%. All materials were supplied by Sigma unless otherwise stated. Adipose tissue was obtained by liposuction from the abdominal, hip and thigh regions of healthy female donors with an average age of 30 (range: 18 to 39). The stromal vascular fraction (SVF) was separated from adipose tissue using a modified procedure as previously described (Zuk, P.A., Zhu, M., Ashjian, P., De Ugarte, D., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P. & Hedrick, M.H. Human adipose tissue is a source of multipotent stem cells. Molec. Biol. Cell. 13, 4279-4295 (2002). Briefly, washed lipoaspirate (300-400 ml) was digested with 0.1% collagenase in HBSS containing antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) and 2.5 g/ml amphotericin B (Gibco) for 2 h on a shaker at 37ºC. Floating adipocytes were aspirated from pelleted SVF after centrifugation at 400g for 10 min. Erythrocytes were eliminated by resuspending SVF pellets in lysis buffer (2.06 g/L Tris base, 7.49 g/L NH4Cl, pH 7.2) for 10 min. Resuspended cells in HBSS were then passed through 100 µm, then 40 µm cell sieves (Falcon). Cell suspensions were then applied to Histopaque-1077 gradients. After centrifugation, adipocyte-derived adult stem cells (ADASCs) at the gradient interface were isolated, washed in HBSS and passed through a 30 µm cell mesh. Cell counts and viability assessment were performed. CD45- cells were negatively selected from the freshly isolated ADASCs using anti-CD45 FITC MicroBeads followed by selection with Super MACS (Milteny Biotec, Bergish Gladbach, Germany), according to the manufactures protocol. CD45-CD31+ and CD45-CD31- ADASCs were separated by using anti-CD31 FITC monoclonal antibody followed by anti-FITC MicroBeads and Super MACS (Milteny Biotec) according to the manufactures protocol. Separation was confirmed using flow cytometry. Immediately after separation, aliquots of each cell population, i.e. CD45-CD31+ and CD45-CD31- ADASCs, were snap frozen in liquid nitrogen and RNA could subsequently be extracted (see extraction protocol). Total RNA was isolated by use of TRIzol reagent (Invitrogen, Life Technologies) according to the manufactures protocol. We started with 100 ng total RNA, using Affymetrix ”Small Sample Labeling Protocol vII”. The ” Small Sample Labeling Protocol vII” contains two cycles of cDNA synthesis and in vitro transcription (IVT). First cycle: cDNA was synthesised by SuperScript II (Invitrogen, Carlsbad , California) and a primer containing a poly(dT) and a T7 RNA polymerase promotor sequence (Affymetrix, Santa Clara, California). cRNA was obtained using Megascript High Yield Transcription Kit (Ambion, Austin, Texas). Second cycle: cDNA was synthesised by SuperScript II and Random Primers (Invitrogen, Carlsbad , California) from 400 ng cRNA. Biotinlabeled cRNA was synthesised from cDNA in the presence of biotinylated UTP and CTP, using ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY). We started with 100 ng total RNA, using Affymetrix ”Small Sample Labeling Protocol vII”. The ”Small Sample Labeling Protocol vII” contains two IVT amplificationsteps (In vitro Transcription). The biotin labeling takes place in the second step using double-stranded cDNA (obtained from 400 ng cRNA) as a template in the presence of biotinylated UTP and CTP. The ENZO BioArray HighYield RNA Transcript Labeling Kit (Affymetrix, Santa Clara, CA, and ENZO Diagnostics, Farmingdale, NY) was used, according to the manufacturer's instructions. Title: Fluidics Station Protocol. Description: Title: Affymetrix CEL analysis. Description: Title: Affymetrix CHP Analysis (ExpressionStat). Description: Protocol Parameters Extracted product;Amplification; Amount of nucleic acid labeled;Label used;Amplification; Protocol Hardware Protocol Software MicroArraySuite 5.0 MicroArraySuite 5.0 MicroArraySuite 5.0 Protocol Contact Protocol Term Source REF mo The MGED Ontology SDRF File E-MEXP-872.sdrf.txt Term Source Name ncbitax atcc NCI_thesaurus nci_thesaurus mo The MGED Ontology ArrayExpress The MGED Ontology mo EFO Term Source File http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html http://www.atcc.org/ ncithesaurus.obo.alt http://nciterms.nci.nih.gov/NCIBrowser/Dictionary.do http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/arrayexpress http://mged.sourceforge.net/ontologies/MGEDontology.php http://mged.sourceforge.net/ontologies/MGEDontology.php http://www.ebi.ac.uk/efo/ Term Source Version