Gene expression changes in mouse hematopoietic stem cell enriched populations following the loss of Meis1 expression
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ABSTRACT: Hematopoietic stem cell enriched Lin-Sca1+cKit+ cells were isolated from heterozygous and homozygous transgenic mice with a loxP-flanked Meis1 allele following in vivo allele deletion.
Project description:The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Our present study suggested it exerts two distinguishable effects in differentiating ES cells. First, it increases the numbers of hematopoietic progenitors and extends their persistence in culture. Second, Meis1 skews hematopoietic differentiation by suppressing erythroid while enhancing megakaryocytic progenitor differentiation. To identify the underlying transcriptional bases of these actions, we carried out microarray analysis to compare the various populations of cells developing in ES differentiation cultures in the presence and absence of Meis1 induction. ES cells with dox-inducible Meis1 (A2lox.Meis1) were differentiated as embryoid bodies (EBs) for 6 days before plating on OP9-GFP cell monolayers and cytokines, and treated with (+) or without (-) doxycycline (dox). Cells were purified by cell sorting on day 7 or 8 into various populations based on levels of CD41 expression: GFP-CD41-, GFP-CD41+ (day 7) and GFP-CD41-,GFP-CD41int, and GFP-CD41hi (day 8). Gene expression of these purified populations was determined by microarray analysis.
Project description:Murine CD45.1+ CD117+ BM cells were co-transduced pairwise with retroviral vectors expressing Hoxa9, Meis1, Foxc1 or a control empty vector. Ninety-six hours later 10^6 drug resistant cells were transplanted into CD45.2+ irradiated congenic recipients. To investigate the consequences of FOXC1 expression on the transcriptome in murine AML, we performed exon array analysis using flow sorted CD117+Gr1+ leukemia cells recovered from sick mice (Hoxa9/control; Moxa9/Meis1; or Hoxa9/FOXC1 leukemias; 3 mice per cohort). Nine exon arrays total; 3 arrays for each cohort (Hoxa9/control; Moxa9/Meis1; or Hoxa9/FOXC1), each from a separate mouse.
Project description:To identify the molecular characterisitics of parallel lineage-biased MPP populations arising from hematopoietic stem cells (HSC) we conducted genome-wide analyses of hematopoietic stem, progenitor and mature myeloid cell populations using Affymetrix Gene ST1.0 arrays. Microarray analysis of 3-5 biological replicates of the indicated hematopoietic populations, isolated by FACS sorting from C57BL/6 mouse BM. Immunophenotypic definitions: Long-term HSC (HSCLT) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+); Short-term HSC (HSCST) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150-); MPP2 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150+); MPP3 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150-); MPP4 (Lin-/cKit+/Sca1+/Flk2+); CMP (Lin-/cKit+/Fc?R-/CD34+); GMP (Lin-/cKit+/Fc?R+/CD34+); Pre-granulocyte (PreGr) (Mac1+/Gr1int); Granulocyte (Gr) (Mac1+/Gr1hi). HSC and GMP samples listed here were also used as controls for our related microarray study GSE48893.
Project description:Expression profiling of FACS purified Lin-cKit+ cells from compound URE-/+::Msh2-/- mice with AML and control animals Analysis of gene expression in Lin-cKit+ cells from three URE-/+::Msh2-/- mice and four wild type controls
Project description:Expression profiling of FACS purified Lin-cKit+ cells from preleukemic compound URE-/+::Msh2-/- mice and control animals (two separate pools of 3 mice each) Analysis of gene expression in Lin-cKit+ cells from two URE-/+::Msh2-/- mice and wild type control animals (two pools of three animals each)
Project description:Mouse haematopoietic stem cells (HSCs) undergo a post-natal transition in several properties, including a marked reduction in their self-renewal activity. We now show that the developmentally timed change in this key function of HSCs is associated with their decreased expression of Lin28b and an accompanying increase in their let-7 microRNA levels. Lentivirus(LV)-mediated overexpression of Lin28 in adult HSCs elevates their self-renewal activity in transplanted irradiated hosts, as does overexpression of Hmga2, a well-established let-7 target that is upregulated in fetal HSCs. Conversely, HSCs from fetal Hmga2-/- mice do not display the heightened self-renewal activity that is characteristic of wild-type fetal HSCs. Interestingly, overexpression of Hmga2 in adult HSCs does not mimic the ability of elevated Lin28 to activate a fetal lymphoid differentiation program. Thus Lin28b may act as a master regulator of developmentally timed changes in HSC programs with Hmga2 serving as its specific downstream modulator of HSC self-renewal potential. Lin-Sca1+cKit+ cells were isolated from E14.5 fetal livers (of wild-type of Hmga2-/- embryos) or the bone marrow of 8-12 week old mice by fluorescence activated cell sorting. The RNA was extracted and hybridized on Affymetrix mpuse gene 1.0 ST microarrays.
Project description:Sca1+/cKitâ hematopoietic BMCs of hosts bearing primary tumors promote the growth of distant tumors that form with a myofibroblast-rich, desmoplastic stroma. BMCs from old mice bearing primary tumors lack this ability Sca1+/cKit- BMCs were isolated from young and old mice bearing Matrigel control or size-matched primary tumors by FACS directly into RLT Plus Buffer (Qiagen Rneasy Plus Micro Kit)
Project description:In the murine HoxA9/Meis1 (H9M) AML model leukemic stem cell (LSC) potential lies in three different heterogeneous immunophenotypes including Lin-cKit+ progenitor cells (Lin-), Gr1+CD11b+cKit+ myeloid cells and lymphoid cells (Lym+). We performed scRNA-seq to reveal the cell type contribution of HoxA9/Meis1 bone marrow and the hierarchical organization of different immunophenotypes. Therefore, primary and secondary bone marrow of HoxA9/Meis1 tranplanted animals were isolated, sorted and sequenced. We could identify that in the HoxA9/Meis1 AML model a pro-B cell population resides at the apex.
Project description:Our testis transplantation data demonstrate that only Sox2-GFP+c-kit- cells contain testis-repopulating potential and we wondered whether a molecular comparison of the Sox2-GFP+c-kit+ and Sox2-GFP+c-kit- spermatogonial cells would uncover genes that could explain the exclusive repopulation potential of the latter cell population. To this end, we sorted individual testis cell populations and subjected extracted and amplified RNA to array analysis. Mouse testes of 2-week old Sox2GFP mice were isolated, and dissociated with collagenase. Single cell suspensions were generated and stained with FACS antibody for ckit. FACS analysis was done based on internal GFP signal and ckit antibody signal. PI was used to exclude dead cells. Sox2GFP+ckit- and Sox2GFP+ckit+ populations were sorted into Trizol and sent to ExpressionAnalysisR for processing and profiling. 20 testis were pooled into one sample, two biological replicates were analyzed.
Project description:We performed gene expression profiling on in vitro derived PGCs, undifferentiated ESCs, and somatic cells from the EB to examine germ cell expression in ESC-derived cells All cells were collected using fluorescence activated cell sorting to isolate SSEA1+/cKit+ ESCs, SSEA1+/cKitbright PGCs, Oct4-gfp+/cKitbright PGCs, and SSEA1-/cKit- somatic cells and Oct4-gfp-/cKit- somatic cells.