The induction of integrin beta3 signaling in hematopoietic stem cells (HSCs)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE33696 | GEO | 2011/11/16
SECONDARY ACCESSION(S): PRJNA148277
REPOSITORIES: GEO
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Project description:To determine the molecules involved in beta3 integrin signaling in HSCs, we carried out DNA microarray analyses using HSCs stimulated with ligand-mimic antibody for intetegrin beta3 in the presence of stem cell factor(SCF) and/or thrombopoietin (TPO). All experiments were performed in duplicate or triplicate using sorted cells that were independently prepared in separate experiments. After CD34-KSL HSCs were cultured with 2C9.G2 or control IgG for 5 days in S-Clone SF-03 medium supplemented with 50 ng/ml SCF and/or 50 ng/ml TPO, gene expression profiles were examimed using 5,000 sorted CD48-KSL cells from each sample.
2011-11-16 | E-GEOD-33696 | biostudies-arrayexpress
Project description:Hematopoietic stem cells (HSCs) are cells able to self-renew and differentiate into all blood lineages. We previously have performed serum-free single-cell culture for HSC1 (CD201+CD150+CD48-CD41-CD34-LSK) and competitive repopulation with cultured cells. Single highly purified HSC1 were cultured with stem cell factor (SCF), SCF + G-CSF, or SCF + thrombopoietin (TPO) for 7 days. SCF directly acted on HSC1 and maintained their long-term multilineage reconstitution after serial transplantation. Compared with SCF alone, SCF+G-CSF neither enhanced the number of divisions of HSC1 nor enhanced their levels of reconstitution after serial transplantation. SCF+TPO significantly increased the number of divisions of HSC1. The levels of reconstitution of each lineage in SCF + TPO culture were significantly increased at early months after primary transplantation, however, it gradually decreased after secondary transplantation. Therefore, we performed single-cell RNA-Seq to examine the gene expression of cells cultured with different cytokine combinations.
2023-06-28 | GSE153430 | GEO
Project description:Eltrombopag, a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy to stimulate sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. While the functional and molecular effects of Eltrombopag on megakaryopoiesis have been carefully studied in the recent past, insights into its mechanistic impact on the earliest stages of hematopoiesis have been limited. Additionally, previous studies also revealed molecular pathway of Eltrombopag apart from stimulation of TPO signaling, detail characterization remains to be addressed. In this study, we investigated the effects of Eltrombopag, in comparison with TPO, on highly-purified primary hematopoietic stem cells (HSCs) from healthy human donors. The binding of Eltrombopag to TPO-R is species-specific to human and primate cells. we also utilized HSCs isolated mouse as separation-of-function models to directly assess the molecular effect of Eltrombopag on HSCs independent of TPOR stimulation.
2018-09-17 | GSE107429 | GEO
Project description:Eltrombopag, a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy to stimulate sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. While the functional and molecular effects of Eltrombopag on megakaryopoiesis have been carefully studied in the recent past, insights into its mechanistic impact on the earliest stages of hematopoiesis have been limited. Additionally, previous studies also revealed molecular pathway of Eltrombopag apart from stimulation of TPO signaling, detail characterization remains to be addressed. In this study, we investigated the effects of Eltrombopag, in comparison with TPO, on highly-purified primary hematopoietic stem cells (HSCs) from healthy human donors. The binding of Eltrombopag to TPO-R is species-specific to human and primate cells. we also utilized HSCs isolated mouse as separation-of-function models to directly assess the molecular effect of Eltrombopag on HSCs independent of TPOR stimulation.
2018-09-17 | GSE107428 | GEO
Project description:Essential Thrombocythemia (ET) patients, as well as normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic alpha2B-beta3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of alpha2B-beta3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA).
2008-06-14 | E-GEOD-567 | biostudies-arrayexpress
Project description:The aim of these experiments was designed to compare gene expression in human myeloma cell lines expressing beta 3 integrin vs counterpart cell lines that do not express beta 3 integrin. Keywords: Gene expression,cell lines, siRNA Phenotype characterization of both primary and cultured myeloma plasma cells was assessed by flow cytometry (FACScanto, Becton Dickinson, San Jose, CA) using a panel of MoAbs or antisera to the following markers: CD138, CD38, CD56, k/l chains, CD20, CD44, CD54, alphav and beta3 chains. The microarray analysis was performed on: myeloma bone resorbing cell lines alphav-beta3 positive and the same cell line silenced for the integrins; myeloma non bone resorbing cell lines negative for the expression of avb3.
2010-05-19 | E-GEOD-15867 | biostudies-arrayexpress
Project description:Purpose: The goals of this study are to elucidate the influence of integrin β3 signaling on STAT1-dependnet gene expression in IFNγ-treated HSCs. Methods: Wild type (WT) HSCs were cultured with or without IFNγ and/or VN in the presence of stem cell factor (SCF) plus thrombopoietin (TPO). Subsequently, cultured HSC fraction (CD48- c-kit+ Sca-1+ Lineage-) were sorted, followed by mRNA sequence using Ion Proton (n>4). Moreover, to extract genes whose expression were changed via STAT1 in the presence of IFNγ, mRNA profiles of STAT1-/- HSCs treated with or without IFNγ were also generated by the same way. The sequence reads that passed quality filters were analyzed by CLC genomic workbench. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm10) with CLC genomic workbench. Indeed, hierarchical clustering analysis showed that IFNγ-treated STAT1-/- HSCs was categorized to the group including Wt HSCs cultured in the absence of IFNγ rather than HSCs treated with IFNγ. Furthermore, gene set enrichment analysis (GSEA) showed that STAT1-dependent upregulated gene sets were significantly enriched within genes whose expression was enhanced in HSCs treated with VN and IFNγ. In contrast, integrin β3 signaling in the absence of IFNγ appears to not influence the expression of IFNγ/STAT1-dependent genes, as evidenced by the observation that VN treatment was statistically and significantly independent of the enrichment of gene sets that were both up-regulated by STAT1 Conclusions: Our study represents that STAT1 plays a central role in IFNγ-mediated HSC responses and integrin β3 signaling in HSCs promotes STAT1-dependent gene expression in the presence of IFNγ.
2017-06-01 | GSE81559 | GEO
Project description:Thrombopoietin (TPO) was shown to prevent irradaition-induced hematopoietic stem cells (HSCs) loss of function. A single injection of TPO to mice 45-60 min prior to irradiation is sufficient to reverse the long-lasting accumulation of persistent DNA damage. We used microarrays to analyze whether this effet can relie on TPO-induced specific transcriptional changes during this early time.
2017-12-31 | GSE84195 | GEO
Project description:Essential Thrombocythemia (ET) patients, as well as normal donors BM CD34+ cells were differentiated in thrombopoietin-treated liquid suspension cultures. Briefly, CD34+ cells (80,000 /mL) were resuspended in a serum-free medium in the presence of 100 ng/mL of human recombinant thrombopoietin (TPO; Genzyme, Boston, MA). Every 3 days, viable cells were scored by Trypan blue dye exclusion and cultures were amplified with fresh serum-free medium. Each well was then supplemented with 100 ng/mL TPO. After 14 to 16 days of liquid culture, CD34-derived megakaryocytes (MKs) were purified by means of an anti-CD41a monoclonal antibody (MoAb) (Dako, Milan, Italy) directed against the glycoproteic alpha2B-beta3 complex and immunobeads (MPC 450 Dynabeads; Dynal, Oslo, Norway). The purity of MKs was determined for each isolation by indirect immunofluorescence, using an anti-CD41b MoAb which reacts with a different epitope of alpha2B-beta3 subunit (Immunotech Inc., Westbrook, ME), followed by a goat anti-mouse IgG, covalently linked to fluorescein (Becton Dickinson, San Jose, CA). Keywords: other
2003-12-25 | GSE567 | GEO
Project description:Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help develop novel therapeutic approaches for hematopoietic disorders.
2023-12-16 | GSE249912 | GEO