Project description:Epigenetic regulation plays an important role in cellular development and differentiation. A detailed map of the DNA methylation dynamics that occur during cell differentiation would contribute to decipher the molecular networks governing cell fate commitment. We used the most recent Illumina MethylationEPIC Beadchip platform to describe the genome-wide DNA methylation changes observed throughout hematopoietic maturation by analyzing multiple hematopoietic cell types at different developmental stages. We identified a plethora of DNA methylation changes that occur during human hematopoietic differentiation. Interestingly, we observed that T lymphocytes display a substantial enhancement of de novo CpG hypermethylation as compared to other hematopoietic cell populations.
Project description:Chronic myelomonocyticleukemia (CMML) is a clinically heterogeneous stem cell malignancy with overlapping features of myelodysplasiaand myeloproliferation. CMML is further subclassified according to percentage of leukemic blasts present in blood or bone marrow, reflecting its intrinsic tendency to progression towards acute myeloid leukemia. Over 90% of CMML patients carry founding mutations in epigenetic and/or splicing genes, which typically involve the primitive stem cell compartment. Thus, transcriptional dysregulation at the stem cell level is likely fundamental to disease onset and progression. However, the critical early hematopoietic stem and progenitor cell (HSPC) subpopulation has not been studied in CMML. We performed single cell RNA sequencing on>6,800 Lin-CD34+CD38-primitive HSPCs from seven CMML patients and three healthy controls. We found substantial inter-and intra-patient heterogeneity, with CMML stem cells displaying distinct transcriptional programs, differentiation potential and cellular signaling pathway priming. Pseudotime analyses revealed that CMML-1/CMML-2 HSPCs have distinct cellular trajectories, indicating that transformation events initiate early within the hematopoietic hierarchy and suggesting against a simple linear clonal evolution dynamic in acute leukemic transformation. We further identified several transcription factors uniquely active in distinct sample subsets. Together our findings provide novel insights into the CMML stem cell compartment, revealing an unexpected degree of transcriptional and subclonal heterogeneity and highlighting early mediators of disease initiation and transformation, of potential translational importance
Project description:Aim of the experiment was to compare the gene expression responses of long-term hematopoietic stem cells (HSCs) and myeloid-restricted progenitors (MyPs) to the hematopoietic cell-restricted deletion of the catalytic subunit Elp3 of the Elongator complex. Since Elp3 deletion resulted in activation of the p53 pathway, we also studied the transcriptome of MyPs deficient for Trp53 or for both Elp3 and Trp53. The Elp3fl/fl strain was generated in house and first described in (DOI: 10.1084/jem.20142288). The Trp53 strain was first described in (DOI: 10.1101/gad.14.8.994) and was purchased from the Jackson Laboratory. Littermates of 8-12 weeks old were used in all experiments.
Project description:While therapeutic angiogenesis holds promise for vascular diseases, progress has been limited due to discrepancies in defining adult endothelial progenitors. We have identified and characterized a population of pluripotent derived NRP1+CD34+ nascent endothelial cells, immediately diverged from NRP1+CD34- mesodermal cells. We contrasted the transcriptional profile of NRP1+ CD34+ nascent endothelial cells against undifferentiated human pluripotent stem cells and human umbilical vein endothelial cells (HUVECs) to gain insights into the pathways that are uniquely activated in formation of new endothelium. We found significant upregulation of IL-6 related growth factor receptors, including ciliary neurotrophic factor receptor (CNTFR), and their downstream JAK/STAT signaling components in NRP1+CD34+ endothelial cells. When exposed to CNTF, angiogenic sprouting of NRP1+CD34+ was induced in Matrigel, which was abolished with JAK2 inhibition. Furthermore, we found evidence of JAK2 dependent cytokine signaling in more mature endothelium, highlighting the significance of the IL6R/JAK2 pathway, well known in hematopoiesis, in vascular biology. The findings identify a novel group of growth factor receptors and downstream signaling components that may be targeted to modulate angiogenesis and vasculogenesis. 7 samples analyzed, 2 replicates for NRP1+CD34+, 3 replicates for NRP1+CD34-
Project description:Tumors contain a fraction of cancer stem cells that maintain the propagation of the disease. The CD34CD38_ cells, isolated from acute myeloid leukemia (AML), were shown to be enriched leukemic stem cells (LSC). We isolated the CD34CD38_ cell fraction from AML and compared their gene expression profiles to the CD34CD38 cell fraction, using microarrays. We found 409 genes that were at least twofold over- or underexpressed between the two cell populations. These include underexpression of DNA repair, signal transduction and cell cycle genes, consistent with the relative quiescence of stem cells, and chromosomal aberrations and mutations of leukemic cells. Comparison of the LSC expression data to that of normal hematopoietic stem cells (HSC) revealed that 34% of the modulated genes are shared by both LSC and HSC, supporting the suggestion that the LSC originated within the HSC progenitors. We focused on the Notch pathway since Jagged-2, a Notch ligand was found to be overexpressed in the LSC samples. We show that DAPT, an inhibitor of gamma-secretase, a protease that is involved in Jagged and Notch signaling, inhibits LSC growth in colony formation assays. Identification of additional genes that regulate LSC self-renewal may provide new targets for therapy. Microarrays were used to compare the gene expression patterns between AML CD34+CD38- cells and AML CD34+CD38+
Project description:Physical factors can have major influences on the proliferation and differentiation fate of hematopoietic stem cells in culture. Recently, we demonstrated that cord blood CD34+ cells undergo accelerated and increased megakaryocyte differentiation when incubated at 39°C. In this study, we investigated in detail the impacts of mild hyperthermia on the kinetics of megakaryocyte differentiation, maturation, polyploidization and cell viability. The qualitative and quantitative effects on Mk differentiation were found to be rapidly induced, and optimization of the culture length at 39°C led to greater Mk yields. Mild hyperthermia did not promote endomitosis of cord blood-derived Mk, but rather led to a small reduction in the proportion of polyploid Mk. Moreover, it had little impact on viability but induced a significant shift in the proportion of cells undergoing apoptosis at 37°C to necrosis at 39°C. Finally, our results suggest that the effects on megakaryocyte differentiation could be the consequences of aberrant expression of key megakaryocyte transcription factors induced by mild hyperthermia. Experiment Overall Design: To define potential differences between megakaryocytes (Mk) derived at 37°C or 39°C, we compared their gene expression repertoire by micro-gene chip technology (Affymetrix GeneChip HG133A). Two independent experiments were carried out on Mk at 37 or 39°C. CD41a+ Mk issued from CB CD34+ cells were cultured for 7-days with TPO at 100 ng/ml and purified by positive magnetic selection using a CD41a-FITC monoclonal antibody (Immunotech, Marseille, France) and MACS columns according to manufacturerâs instructions (Miltenyi, Auburn, CA, USA) with a purity of >95%.
Project description:Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application towards dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune cells in mouse tumors and identified specific macrophage and conventional dendritic cell (cDC) subsets that are comparable to previously described human myeloid populations. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R selectively depleted macrophages with an inflammatory signature but spared a macrophage population that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated of a cDC1-Ccl22 population and gradually increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing. Raw FASTQ sequences have been uploaded to European Nucleotide Archive (Study #: PRJEB34105 (ERP116961))
Project description:Chronic lymphocytic leukemia (CLL), the most frequent adult leukemia in western countries, is a clonal accumulation of mature B-lymphocytes and its natural history is yet unclear. By using sequencing and cellular biology approaches on a cohort of CLL patient samples, we show here that acquired CLL mutations are observed in hematopoietic multipotent progenitor fractions in the majority of patients. These early CLL mutations include recurrent inactivating mutations in NFKBIE (10.7%) and missense mutations in BRAF (3.6%) and EGR2 (8.3%). Functional analyses demonstrated that BRAF-G469R affects lymphoid differentiation and transforms the T-cell lineage in vivo. In addition, the EGR2 recurrent mutations were associated with transcriptional activation of EGR2 target genes in patients and cell cycle abnormality in cellular model. Our findings indicate that CLL may develop from an initial infra-clinic, pre-leukemic phase affecting immature hematopoietic cells. The aim of this study is to compare exome sequences from tumor cells and T-lymphocytes in order to predict somatic mutations in 24 CLL patients (17 IGHV-unmutated and 7 IGHV-mutated). Enriched exome fragments were subjected to massively parallel sequencing using HiSeq 2000 (Illumina).
Project description:Therapy-related myelodysplasia or acute myeloid leukemia (t-MDS/AML) is a lethal complication of cancer treatment. Although t-MDS/AML development is associated with known genotoxic exposures, its pathogenesis is not well understood and methods to predict risk of development of t-MDS/AML in individual cancer survivors are not available. We performed microarray analysis of gene expression in samples from patients who developed t-MDS/AML after autologous hematopoietic cell transplantation (aHCT) for Hodgkin lymphoma (HL) or non-Hodgkin lymphoma (NHL) and controls that did not develop t-MDS/AML after aHCT. CD34+ progenitor cells from peripheral blood stem cell (PBSC) samples obtained pre-aHCT from t-MDS/AML cases and matched controls, and bone marrow (BM) samples obtained at time of development of t-MDS/AML, were studied. Significant differences in gene expression were seen in PBSC obtained pre-aHCT from patients who subsequently developed t-MDS/AML compared to controls. Genetic alterations in pre-aHCT samples were related to mitochondrial function, protein synthesis, metabolic regulation and hematopoietic regulation. Progression to overt t-MDS/AML was associated with additional alterations in DNA repair and DNA-damage checkpoint genes. Altered gene expression in PBSC samples were validated in an independent group of patients. An optimal 63-gene PBSC classifier derived from the training set accurately distinguished patients who did or did not develop t-MDS/AML in the independent test set. These results indicate that genetic programs associated with t-MDS/AML are perturbed long before disease onset, and can accurately identify those at risk of developing this complication. PBSC samples obtained pre-aHCT and BM samples at the time of development of t-MDS/AML post-HCT were studied. The training set consisted of 18 patients who developed t-MDS/AML (M-bM-^@M-^]casesM-bM-^@M-^]) after aHCT, matched with 37 controls who underwent aHCT, but did not develop t-MDS/AML. One to three controls were selected per case, matched for primary diagnosis (HL/NHL), age at aHCT (M-BM-110years), and ethnicity (Caucasians, African-Americans, Hispanics, other). The length of follow-up after aHCT for controls was longer than the time to t-MDS/AML in the corresponding case. The results of the training set were validated in an independent group of 36 patients (test set) consisting of 16 cases that developed t-MDS/AML post-aHCT and 20 matched controls. In the test set, 55 PBSC samples from 18 cases and 37 matched controls were studied. BM samples from time of development of t-MDS/AML were available for 12 cases, and from 21 matched controls obtained at a comparable time from aHCT. For validation, 36 PBSC samples from 16 cases and 20 matched controls were studied. All samples had been cryopreserved as mononuclear cells. After thawing, samples were labeled with anti-CD34-APC and anti-CD45-FITC and CD34+CD45dim cells were selected using flow cytometry. Total RNA was extracted using the RNeasy kit. RNA from 1000 cells was amplified and labeled using GeneChipM-BM-. Two-Cycle Target Labeling and Control Reagents from Affymetrix. 15 M-BM-5g of cRNA each was hybridized to Affymetrix HG U133 plus 2.0 Arrays. Microarray data were analyzed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). Data for PBSC and BM samples were normalized separately using robust multiarray averages with consideration of GC content (GCRMA). Probesets with low expression or variability were filtered. Expression of genes represented by multiple probesets was set as the median of the probesets. Using conditional logistic model (CLM) to retain matching between cases and controls, we analyzed the magnitude of association [expressed as odds ratio (OR)] between t-MDS/AML and i) gene expression levels in PBSC at the pre-aHCT time point; ii) gene expression levels in BM at time of t-MDS/AML; and iii) change of expression of individual genes from PBSC to time of t-MDS/AML. False discovery rate (FDR) was applied to adjust for multiple testing. Gene set enrichment analysis (GSEA) was performed on ranked lists of genes differentially expressed between cases and controls. Where multiple significant gene sets were related to each other, analysis was performed to identify a subset of common enriched genes. Average gene expression was calculated for each set and heatmaps plotted to show the contrasts between cases and controls. Gene Ontology (GO) and pathway analysis was performed using DAVID 2008 and Ingenuity IPA 7.5 respectively, retaining genes with z-scores M-bM-^IM-%1.8 or M-bM-^IM-$-1.8, and M-bM-^IM-%1.5-fold change in OR between cases and controls. The association between gene expression in the PBSC product and subsequent development of t-MDS/AML identified in the training set was validated in an independent test set of 36 PBSC sample procured from patients who developed t-MDS/AML after aHCT (16 cases) or did not (20 controls). Pre-processing, normalization and filtering procedures for the test set were identical to the training set. Differential expression between cases and controls was analyzed using CLM. GSEA analysis was performed on the ranked list of differentially expressed genes. Prediction analysis of microarray (PAM) was used to derive a prognostic gene signature from the training set to classify patients as case or control. PAM uses the M-bM-^@M-^\nearest shrunken centroidM-bM-^@M-^] approach and 10-fold cross-validation to select a parsimonious gene expression signature that can classify samples with minimal misclassification. PAM was applied to genes common to both datasets. Based on the misclassification error in cross-validation, a 63-gene signature was selected for prediction using the test data.