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Temporal expression profile of megakaryocytic differentiation primary CD34+ cell culture

ABSTRACT: Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of Mk differentiation human CD34-positive hematopoietic stem and progenitor cells cultured with thrombopoietin, interleukin-3, and Flt3-ligand. The goal this study was to identify genes involved in the various facets of Mk differentiation including commitment, polyploidization, proplatelet formation, and apoptosis. Keywords: time course

ORGANISM(S): Homo sapiens

PROVIDER: GSE3839 | GEO | 2006/11/08

SECONDARY ACCESSION(S): PRJNA104987

REPOSITORIES: GEO

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Transcription profiling of human megakaryocytic differentiation primary CD34+ cell culture

Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of Mk differentiation human CD34-positive hematopoietic stem and progenitor cells cultured with thrombopoietin, interleukin-3, and Flt3-ligand. The goal this study was to identify genes involved in the various facets of Mk differentiation including commitment, polyploidization, proplatelet formation, and apoptosis. Experiment Overall Design: G-CSF mobilized peripheral blood CD34-positive cells were cultured with TPO, IL-3, and Flt3-L to induce Mk differentiation. Samples prior to day 5, including uncultured starting cells, were analyzed directly, whereas samples after and including day 5 were positively selected for CD41a expression immediately prior to RNA isolation. Three biological replicate experiments were analyzed and approximately one-half of the samples from each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.

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Temporal expression profile of CHRF-288 cell line after phorbol ester stimulation

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Transcription profiling of human CHRF-288 cell line after phorbol ester stimulation

Project description:Little is known about the global transcriptional program underlying megakaryocytic (Mk) differentiation, maturation, and apoptosis. Using DNA microarrays and Q-RT-PCR, we examined the transcriptional profile of phorbol-ester-induced Mk differentiation of the megakaryoblastic CHRF-288-11 (CHRF) cell line â a model system for investigating megakaryopoiesis. The goals of this study were to (1) verify the megakaryocytic nature of the CHRF cell line at the transcriptional level, and (2) extract novel insights into the key facets of Mk maturation including polyploidization, proplatelet formation, and apoptosis. Experiment Overall Design: CHRF-288 cells were cultured in the presence of 10 ng/mL phorbol ester (PMA) or equivalent volume of DMSO solvent (0.02%). Unstimulated control cells from time zero (exponentially growing CHRF cells) were also analyzed. For PMA treated legs, only the adherent cells were included in the transcriptional analysis. Two biological replicate experiments were analyzed and approximately one-half of the samples with each experiment were technically replicated. Hybridizations were performed in a reference design with all samples labeled with Cy3 and a reference RNA pool labeled with Cy5.

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thrombopoietin-induced megakaryocyte differentiation with and without nicotinamide

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Transcription of human thrombopoietin-induced megakaryocyte differentiation with and without nicotinamide vs. Universal reference sample

Project description:Culture of hematopoietic stem and progenitor cells in the presence of thrombopoietin induces megakaryocytic differentiation. Addition of nicotinamide to thrombopoietin-stimulated cultures results in significant increases in megkaryocytic differentiation including greater polyploidization and enhanced proplatelet formation. This study focuses on understanding the differences in the temporal gene expression pattern during differentiation with and without nicotinamide. Nicotinamide was added on day 5. Experiment Overall Design: Two biological experiments were analyzed. For each experiment, a sample was taken on day 5, before nicotinamide addition, and on days 6, 8, and 10 from both nicotinamide and control treated cultures. Each sample was analyzed in duplicate for a total of 14 hybridizations per culture or 28 total hybridizations.

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Project description:In order to examine genes whose expression is altered as a result of the knock-down (KD) of p53, we used gene expression microarray analysis based on the CHRF cell line, a human megakaryoblastic cell line, which upon PMA stimulation undergoes polyploidization, and extends proplatelet-like cytoplasmic protrusions combined with apoptosis. The CHRF cell line has been validated in our laboratory as a good model of in vitro megakaryopoiesis by comparison to hematopoietic stem cells undergoing Mk differentiation. As described, we have previously generated stable p53-KD CHRF cell lines by lentiviral delivery of microRNA-adapted-short hairpin sequences targeting p53 as well as a suitable scrambled-control cell line.

2011-07-27 | GSE30984 | GEO

MKL1 promotes megakaryocytic differentiation in HEL cells

Project description:Megakaryoblastic Leukemia 1 (MKL1) was identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, but nothing is known regarding its role in hematopoiesis. Here we show that overexpression of MKL1 enhances megakaryocytic differentiation of the Human Erythroleukemia cell line (HEL). Microarray analysis reveals that MKL1 promotes expression of megakaryocyte-specific genes such as glycoprotein V (GP5), as well as cytoskeletal and adhesion molecule genes relevant to megakaryocyte differentiation and proplatelet formation. MKL1 is a transcriptional coactivator of Serum Response Factor. In this study, MKL1 also upregulates known SRF targets. Results provide insight into the role of MKL1 in megakaryocytopoiesis.

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