Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from different stages of hematopoietic cells development

ABSTRACT: 18 different population of cells in different developmental stages in hematopoietic hierarchy have been purifyed by FACS analyses from wild type C57Bl6 mice and subjected to Micrroarray Affymetrix mouse 430.2 platform We used microarrays to compare global expression pattern in different hematopoietic cell populations to reconstruct the whole hierarchy of hematopoietic tree based on gene profiling Keywords: Hematopoietic Development For analysis of progenitor populations, RNA was extracted from 2000 purified adult BM cells using RNAeasy microkit. RNA was labeled and amplified by dual amplification and hybridized to Affymetrix micro array u480A, according to AffymetrixTM GeneChip Expression Analysis Technical Manual. Probe level expression values were calculated using RMA

ORGANISM(S): Mus musculus

SUBMITTER: Sasan Zandi

PROVIDER: E-GEOD-14833 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The major myeloid blood cell lineages, including erythrocytes, platelets, granulocytes and macrophages, are generated from hematopoietic stem cells (HSC) by differentiation through a series of increasingly more committed progenitor cells. Precise phenotypic identification and functional characterization of such intermediate progenitors has important consequences for understanding fundamental differentiation processes and is clinically relevant since such events become dysregulated in various disease settings, including leukemia. While previous studies have suggested a hierarchy for myeloid differentiation involving a common progenitor through which all myeloid lineages are derived, several recent studies have suggested that such a developmental intermediate might not be an absolute requirement. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin and CD41. Our studies reveal a complex hierarchy of myeloerythroid progenitors with distinct and developmentally restricted lineage potentials. Global gene expression signatures of these cellular subsets revealed expression patterns consistent with their functional capacities, while hierarchical clustering analysis provides details on their lineage relationships. These data challenge existing models of hematopoietic differentiation, by suggesting that progenitors of the innate and adaptive immune system in the adult separate late, and to a large extent, following the divergence of megakaryocytic/erythroid potential. Experiment Overall Design: RNA was extracted from purified adult BM subsets using RNeasy mRNA purification kit (Qiagen). Subsequent handling was performed at the SweGene Affymetrix unit at Lund University (http://www.swegene.org/microarray). Briefly, RNA (arrays in triplicate for all progenitor fractions except for pre MegE for which 5 arrays were produced) was labeled and amplified according to AffymetrixTM; Small Sample Labelling Protocol v.2, with the exception that the second round of in vitro transcription (IVT) was performed using AffymetrixTM GeneChipTM Expression 3â??amplification kit. Hybridization and washing was performed according to AffymetrixTM GeneChipTM Expression analysis technical manual. Chips were scanned using an AffymetrixTM GeneChipTM Scanner 3000 and scaled to a median intensity of 100. For subsequent analysis, probe level expression values were extracted using RMA (Irizarry et al., 2003) and subsequent analyses was performed using dChip software (http://biosun1.harvard.edu/complab/dchip/) following filtering (0.5<SD/mean<1,000).

Project description:The homeobox (Hox) transcription factor HOXA10 has been implicated in regulation of hematopoietic cell fate. Here, using a transgenic mouse model where expression of HOXA10 is tightly regulated in a graded, doxycyclin-dependent manner we demonstrate that several key commitment steps in hematopoietic differentiation have distinctly different outcomes depending on the expression level of HOXA10. Similarly, HOXA10 regulates hematopoietic stem cell (HSC) proliferation in a dose dependent manner, since intermediate levels of HOXA10 generated a 4.5-fold increase in long-term repopulating capacity after 13 days of liquid culture, whereas high levels reduced proliferation of HSCs. Interestingly, the effects on HSC proliferation were associated with altered expression of several known regulators of stem cell self-renewal. Taken together, our findings reveal entirely novel functional and molecular aspects of HOXA10 in regulation of hematopoiesis and emphasize the need for tightly regulated production of HOX proteins in possible future applications of stem cell expansion. LSK cells were sorted from rtTA-HA10 and cultured for 72 hours in complete serum free medium in three different concentrations of doxycycline (0, 0.1 and 0.5 µg/ml doxycycline). RNA was extracted using RNeasy Mini kit, Qiagen, labeled and amplified according to AffymetrixTM Two Cycle Target Labeling Protocol. Hybridization and washing was performed according to AffymetrixTM GeneChip Expression protocol. Chips were scanned using an AffymetrixTM GeneChip Scanner 3000. The Affymetrix software GCOS was used to generate cell intensity data files (CEL) from two independent experiments. The CEL file data was then imported into the GeneSpring® software version 7.2 (Silicon Genetics, Redwood City, CA). The GC-RMA method was used for normalization and data was processed as follows: values below 0.01 were set to 0.01. All of the genes in each sample were divided by the median of the specified list of 100 positive control genes present on the MOE430 v2 chip. All samples were then normalized against the median of the untreated control samples. Each measurement for each gene in the treated samples was divided by the median of that gene's measurements in the corresponding control samples. We judged genes to be differentially expressed when the difference in expression between two conditions was at least 2-fold.

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Expression data from different stages of hematopoietic cells development

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