JAK2V617F mutation in CD34+ stem cells of essential thrombocythemia
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Rossella Manfredini
PROVIDER: E-GEOD-9827 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Primary mielofibrosis (PMF) is a rare chronic myeloproliferative disorder characterized by the accumulation of abnormal megakaryocytes (Mks) in the bone marrow (BM), variable degrees of BM fibrosis, osteosclerosis and angiogenesis, immature myeloid and erythroid cells, and tear-drop erythrocytes in the peripheral blood (PB), and extramedullary hematopoiesis. The identification of the JAK2V617F mutation represented a seminal discovery in the field of Philadelphia-chromosome–negative chronic myeloproliferative neoplasms (MPNs), providing clues to the pathogenesis, prompting a revision of the diagnostic criteria, and culminating in the development of clinical trials with JAK2 (and JAK1) inhibitors. The JAK2V617F mutation occurs in almost all patients with polycythemia vera (PV) and in 50%-70% of those with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Soon after the identification of the JAK2V617F mutation, mutations in JAK2 exon 12 were described in rare patients with JAK2V617F-negative PV and mutations in MPL were reported in 5%-10% of ET or PMF subjects. The complexity of the molecular pathogenesis of MPNs is reinforced by discovery of additional mutations in TET2, ASXL1, CBL, IDH1/IDH2, EZH2 and IKZF1. These mutations are detected in a minority of patients at different phases of the disorder, including leukemic transformation, and are variably associated each other and with JAK2 or MPL mutations. In order to better characterize biological differences between mutated and wild-type PMF cell populations we performed a gene expression profiling on 9 samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes and 11 wild-type samples using the Affymetrix GeneChip technology. After data preprocessing and filtering a supervised analysis approach was used to define a gene expression signature for mutated samples. PMF samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes exhibit a specific molecular signature as compared with WT samples. Gene expression profile (GEP) of CD34+ cells from 20 PMF patients (1 replicate for each sample). In particular, GEP was performed on 9 samples carrying at least one mutation in ASXL1, SRSF2 or EZH2 genes and 11 wild-type samples.
2013-07-23 | E-GEOD-41812 | biostudies-arrayexpress
Project description:We show the molecular and functional characterization of a novel population of lineage-negative CD34-negative (Lin- CD34-) hematopoietic stem cells (HSCs) from chronic myelogenous leukemia (CML) patients at diagnosis. Molecular caryotyping and quantitative analysis of BCR/ABL transcript demonstrated that about one third of CD34- was leukemic. CML CD34- cells showed kinetic quiescence and limited clonogenic capacity. However, stroma-dependent cultures and cytokines induced CD34 expression on some HSCs, cell cycling, acquisition of clonogenic activity and increased expression of BCR/ABL transcript. CML CD34- cells showed an engraftment rate in immunodeficient mice similar to that of CD34+ cells. Gene expression profiling revealed the down-regulation of cell cycle arrest genes together with genes involved in antigen presentation and processing, while the expression of angiogenic factors was strongly up-regulated when compared to normal counterparts. Flow cytometry analysis confirmed the significant down-regulation of HLA class I and II molecules in CML CD34-cells. Increasing doses of imatinib mesilate (IM) did not affect fusion transcript levels, BCR-ABL kinase activity and the clonogenic efficiency of CML CD34- cells as compared to leukemic CD34+cells. Thus, we identified in CML a novel CD34- leukemic stem cell subset with peculiar molecular and functional characteristics which may be a potential target for CML therapeutics. Leukemic cells were obtained from 12 chronic phase Ph+ CML patients at diagnosis and before treatment. Normal samples were leukapheresis products from 12 healthy stem cell donors receiving recombinant human granulocyte colony-stimulating factor (G-CSF; Lenograstim, Sanofi-Aventis, Milan, Italy). The protocol was approved by the ethical committee of the University Hospital and each patient/donor gave written informed consent. Hemopoietic stem/progenitor cell purification and phenotypic analyses were performed as previously described (Lemoli et al, Br J Haematol, 2003; Lemoli RM et al., Blood, 1997). Aliquots of sorted Lin-CD34-, Lin-CD34+ and Lin+CD34+ were reanalyzed by FacScan (Becton Dickinson, Franklin Lakes, NJ) to assess their purities. Total cellular RNA was extracted from 0.5x105 cells of each sample using RNeasy Micro kit (Qiagen, Valencia, CA) following the protocol supplied by the manufacturer. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit, Agilent Technologies, Waldbrunn, Germany) were used to determine the concentration and purity/integrity of RNA samples using Agilent 2100 Bioanalyzer. RNAs originating from 12 normal donors or from 12 CML patients were pooled in order to obtain at least 2 mg per sample. One-cycle target labeling assays, as well as the Affymetrix Human HG-U95Av2 GeneChip arrays hybridization, staining, and scanning, were performed, using Affymetrix standard protocols (Affymetrix, Santa Clara,CA).
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Project description:The BM-derived CD45+/Sca1+ cells are haematopoietic stem/progenitor cells that have the ability to circulate and migrate and engraft to the muscle tissue, and therefore they are of particular interest. Notably, these cells retain their haematopoietic potential, as revealed both by in vitro and in vivo assays; but they also acquire myogenic potential, as shown by their ability to participate in muscle regeneration. Whether, this latter remarkable ability is the result of the reprogramming of the BM-CD45+/Sca1+ cells and the activation of a myogenic molecular program within these cells, remains controversial. This study aims to clarify this aspect of the process, investigating the role of the muscle microenviroment and key myogenic transcription factors. Keywords: CD45+/Sca1+ cells, BM, muscle CD45+/Sca1+ cells isolated from the BM or the muscle were processed fresh and their RNA was extracted. Moreover, CD45+/Sca1+ cells isolated from the muscle of BM transplanted or untransplanted mice after injury with Cardiotoxin were processed fresh and their RNA was extracted.
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Project description:Nucleotides triphosphates are extracellular messengers binding to specific plasma membrane receptors (P2Rs) that modulate responses as different as proliferation, differentiation, migration or cell death on several cell types including hematopoietic stem cells. Little and controversial information is available on the role of extracellular nucleotides in human mesenchimal stem cells (hMSCs). In this study, we assessed whether P2Rs are expressed and functional in bone marrow-derived hMSCs. Our results demonstrated, at the mRNA and protein level, the expression of all P2X and P2Y receptor subtypes identified so far. P2R activation by their natural ligands adenosine triphosphate (ATP) and uridine triphosphate (UTP) induced in hMSCs, intracellular Ca2+ concentration changes, plasma membrane depolarization and permeabilization. hMSCs were resistant to the cytotoxic effects of high dose ATP despite the expression of permeabilizing P2Rs as demonstrated by the lack of morphological changes, significant release of intracellular markers of cell death or modification of the mitochondrial network. Gene expression profiling revealed the down-regulation of cell proliferation genes whereas genes involved in cell migration and cytokine production were strongly up-regulated by ATP. Functional studies confirmed the inhibitory activity of ATP on proliferation of hMSCs and clonogenic progenitors. Moreover, ATP exerted a chemotactic effect on hMSCs and increased their migration in response to the chemokine CXCL12. Finally, whereas ATP did not affect T-cell inhibitory activity of hMSCs, the nucleotide increased the production of pro-inflammatory cytokines by hMSCs. Thus, our data show that purinergic signaling modulates hMSC functions and point to a role for extracellular nucleotides on hMSCs biology. hMSCs from 6 healthy donors were seeded at a density of 2.5 x 103 cells/cm2 for 24 hours with or without 1mM ATP. We then assessed the transcriptome profile of ATPM-bM-^@M-^Streated and untreated cells using Affymetrix HG-U133 Plus 2 GeneChip array.
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