Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human hematopoietic stem cells vs. differentiated progeny

ABSTRACT: Hematopoietic stem cells (HSC) continuously regenerate a complete hematologic and immune system. Very few genes that regulate this process have yet been identified. In order to identify factors governing differentiation, we have compared the transcriptome of highly purified HSC with their differentiated progeny, including erythrocytes, granulocytes, monocytes, NK cells, activated and naÃ¯ve T-cells, and B-cells. Chromosomal analysis revealed that HSC were more transcriptionally active than other cell types across most chromosomes. Each lineage expressed ~100 to 400 genes uniquely, including many previously uncharacterized genes. Overexpression of two fingerprint genes resulted in a significant bias in differentiation indicating a role in cell fate determination, demonstrating the utility of these data for modulation of specific cell types. Experiment Overall Design: Goal - Expression profiling of hematopoietic cells Experiment Overall Design: Description - Biological replicates of 10 hematopoietic cell types from female C57Bl/6 mice were purified for microarray analysis Experiment Overall Design: Experimental Factors: wild-type female C57Bl/6 CD45.1 Mus musculus Experiment Overall Design: Experimental Design: 25,000- 50,000 cells were FACS sorted, mRNA was purified by RNAqueous (Ambion), DNAse treated with DNAse1, phenol:chloroform:isoamyl alcohol extracted, and amplified through two rounds of in vitro transcription using MessageAmp (Ambion). Second round was biotinylated with bio-CTP and bio-UTP. Experiment Overall Design: Microarrays: Oligonucleotide, Affymetrix, Moe430 v 2.0 Experiment Overall Design: QC: Biological replicates, correlation coefficient > .95; scale factor <10; 5'/3' ratio < 20

ORGANISM(S): Mus musculus

SUBMITTER: Nathan Boles

PROVIDER: E-GEOD-6506 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Stem cells (SC) exhibit a unique capacity for self-renewal in an undifferentiated state. It is unclear whether the self-renewal of pluripotent embryonic SC (ESC) and of tissue-specific adult SC such as hematopoietic SC (HSC) is controlled by common mechanisms. The deletion of transcription factor Zfx impaired the self-renewal but not the differentiation capacity of murine ESC; conversely, Zfx overexpression facilitated ESC self-renewal by opposing differentiation. Furthermore, Zfx deletion abolished the maintenance of adult bone marrow HSC, but did not affect erythromyeloid progenitors or fetal HSC. In both ESC and HSC, Zfx activated a common set of direct target genes. In addition, the loss of Zfx resulted in the induction of immediate-early and/or stress-inducible genes in both SC types but not in their differentiated progeny. These studies identify the first shared transcriptional regulator of ESC and HSC, suggesting a common molecular basis of self-renewal in embryonic and adult SC. Experiment Overall Design: Global gene expression analysis Experiment Overall Design: For ESC, Zfxnull and control Zfxflox clones were grown on gelatin in medium with Experiment Overall Design: 15% Knockout SR and LIF. For HSC, BM cells were pooled from 4-6 Mx1-Cre+ Zfxflox/y CKO and control Zfxflox/y Cre- littermates 4 days after the last pIÂ·C injection, and Lin- Sca-1+ c-Kit+ cells were flow-sorted. RNA was amplified, labeled and hybridized to Mouse Genome 430 2.0 microarrays containing probes for ~37,000 genes (Affymetrix). Experiment Overall Design: Total RNA (2.5 ug) was isolated from ESC using RNeasy Mini Kit (QIAGEN) and Experiment Overall Design: used for reverse transcription and antisense RNA (aRNA) amplification using Experiment Overall Design: MessageAmp II system (Ambion). The resulting aRNA was labeled with Biotin-16-UTP (Roche Applied Sciences) and Biotin-11-CTP (PerkinElmer Life Sciences) in a single round of linear amplification. Labeled aRNA (average size ~1500 nucleotides) was fragmented in RNA Fragmentation Buffer (QIAGEN) prior to hybridization. Experiment Overall Design: For HSC, pooled BM cells were stained with a biotinylated lineage antibody Experiment Overall Design: cocktail, and Lin+ cells were depleted using streptavidin-conjugated magnetic beads and LS selection columns (Miltenyi Biotech). Enriched cells were stained for lineage, c-Kit and Sca-1, and 5 x 104 LSK cells were sorted directly into Trizol Reagent (Invitrogen). Total RNA was isolated and quantified using the Quant-IT RNA Assay Kit (Invitrogen), and 100 ng of each RNA sample were used for reverse transcription and two-round linear aRNA amplification. The aRNA was amplified without labeling in the first round, and then labeled with Biotin-16-UTP/Biotin-11-CTP in the second round. Labeled aRNA (average size ~800 nucleotides) was fragmented as above. Experiment Overall Design: Labeled RNA samples (10 ug/array) were hybridized to Mouse Genome 430 2.0 Experiment Overall Design: arrays (Affymetrix) at the Columbia University Microarray Project core facility according to the manufacturer's instructions. Samples were hybridized in duplicates, with the correlation between duplicate samples calculated at 0.995-0.997. Quality control and normalization were performed using positive and negative hybridization controls (Affymetrix) spiked into the RNA prior to labeling. Linear amplification of RNA was confirmed for every sample. Array scanning and raw data processing were done using GCOS 1.4 software (Affymetrix).

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Transcription profiling of human hematopoietic stem cells vs. differentiated progeny

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