Project description:DNA methylation analysis on purified human long-term and short-term hematopoietic stem cells (LT-HSC, ST-HSC), common myeloid and megakaryocyte-erythrocyte progenitor cells (CMP, MEP) using HELP arrays. FACS-purified hematopoietic stem and progenitor cell (HSPC) subsets were analyzed for changes in DNA methylation using NimbleGen HELP microarrays. Analysis of DNA methylation of bone marrow-derived HSPC subsets of healthy human donors.
Project description:RNA expression analysis on purified human long-term and short-term hematopoietic stem cells (LT-HSC, ST-HSC), common myeloid and megakaryocyte-erythrocyte progenitor cells (CMP, MEP) using microarrays. FACS-purified hematopoietic stem and progenitor cell (HSPC) subsets were analyzed for changes in RNA expression using NimbleGen gene expression microarrays. Analysis of RNA expression of bone marrow-derived HSPC subsets of healthy human donors.
Project description:Hematopoietic stem cells are both necessary and sufficient to sustain the complete blood system of vertebrates. Here we show that Nfix, a member of the nuclear factor I (Nfi) family of transcription factors, is highly expressed by hematopoietic stem and progenitor cells (HSPC) of murine adult bone marrow. Although shRNA mediated knockdown of Nfix expression in Lineage-Sca-1+c-Kit+ HSPC had no effect on in vitro cell growth or viability, Nfix-depleted HSPC displayed a significant loss of colony forming potential, as well as short- and long-term in vivo hematopoietic repopulating activity. Analysis of recipient mice 4-20 days post-transplant revealed that Nfix-depleted HSPC establish in the bone marrow but fail to persist due to increased apoptotic cell death. Gene expression profiling of Nfix-depleted HSPC reveals that loss of Nfix expression in HSPC is concomitant with a decrease in the expression of multiple genes known to be important for HSPC survival, such as Erg, Mecom, Mpl and Prdm16. These data reveal that Nfix is a novel regulator of HSPC survival post-transplantation and establish, for the first time, a role for Nfi genes in the regulation of this cellular compartment. 3 NFIX depleted samples are compared to 3 wt samples
Project description:Purpose: The goals of this study are to compare the transcriptome profile of mouse hematopoietic stem/progenitor cells (HSPC) from the bone marrow overexpressing the innate immune adaptor protein TIRAP to control vector expressing HSPC using RNA-Seq. Methods: mRNA profiles of wild-type (WT) mice bone marrow overexpressing TIRAP or control vectors were generated by deep sequencing, in triplicate, using the Illumina platform. RNA-Seq data were aligned using JAGuaR (v2.0.3), using the mm10 reference. Expression quantification was performed with Sailfish (v0.6.2), using RefSeq gene models. The differential expression analysis between TIRAP expressing bone marrow and control was performed using DESeq2 (v1.10.1). Results: Using an optimized data analysis workflow, we mapped approximately 92% sequenced reads on average per sample to the mouse genome (build mm10).
Project description:TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) Myelodysplastic syndrome (MDS). Hematopoietic-specific deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cells (HSPC) proportions, altered myeloid differentiation, and progressive cytopenia. A subset of mice transplanted with Tifab knockout (KO) hematopoietic cells develop a bone marrow failure (BMF)-like disease with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPC are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic HSPC defect. Gene expression analysis of Tifab KO HSPC identified dysregulation of immune-related signatures, and hypersensitivity to Toll-like receptor 4 (TLR4) stimulation. TIFAB also forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling in HSPC, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction in vivo. Re-expression of TIFAB in human del(5q) leukemic cells results in attenuated TLR4 signaling and reduced cell viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPC by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML. We performed an expression analysis on sorted lineage-Sca1+cKit+ (LSK) isolated from bone marrow (BM) of 3 month old mice transplanted with Tifab+/+ (WT) or Tifab-/- (KO) BM cells (n = 3 mice/group). We selected this time point to capture the gene expression profile of Tifab-/- LSK after engraftment but prior to overt hematopoietic failure. Total RNA was extracted, purified, reverse transcribed, labeled, and hybridized onto the GeneChip MoGene 2.0 ST Array (Affymetrix). Comparison comprises mRNA expression profile of Tifab+/+ LSK vs. Tifab-/- LSK.
Project description:Bone marrow Hdc-GFP+/hiCD11b+Gr1lo vs Hdc-GFP+/hiCD11b+Gr1hi myeloid cells were isolated from bones from histidine decarboxylase (Hdc) green fluorescent protein (Hdc-GFP) mice. Hdc-GFP+/hiCD11b+Gr1hi cells and Hdc-GFP+/hiCD11b+Gr1/lo cells were sorted by combinations of GFP and myeloid cell surface markers CD11b and Gr1 and their differential mRNA expression compared with Affymetrix microarrays.
Project description:Study of hematopoietic stem and progenitor cell (HSPC) hierarchies in bone marrow cells from myelodysplastic syndromes through the transcriptional analysis of single cells in distinct differentiation states
Project description:Differentially expressed genes of CD11b+Gr-1+ immature myeloid cells (IMCs) in the bone marrow and colonic tumor setting of histidine decarboxylase (HDC)-KO mice were examined by microarray (Affymetrix Mouse 430.2 array). Myeloid differentiation-related candidate genes were sought to be isolated and functionally studied. Total RNA of HDC-expressing CD11b+Gr-1+ IMCs of bone marrow were extracted from HDC-EGFP and HDC-EGFP/HDC-KO mice (3 mice in each group). CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) of colon tumor were sorted from 10-12 colon tumors of WT and HDC-KO mice (5 mice in each group), and pooled to extract total RNA for microarray studies. Two technical replicates for each of the four groups. Four sets of comparisons were performed to screen for upregulated or downregulated genes in the HDC-KO CD11b+Gr-1+ IMCs or MDSCs (experiment group) compared to the WT group: (1) HDC-expressing CD11b+Gr-1+ IMCs of bone marrow of HDC KO mice compared to bone marrow IMCs of WT mice; (2) CD11b+Gr-1+ MDSCs in tumors of HDC-KO mice compared to WT mice; (3) CD11b+Gr-1+ MDSCs of WT colon tumors compared to IMCs in the WT bone marrow; and (4) CD11b+Gr-1+ MDSCs of colon tumors of HDC-KO mice compared to IMCs in the bone marrow of HDC-KO mice.