Project description:This collection contains microRNA expression profiling data for samples purified from umbilical cord blood, belonging to one of the followiing populations: MEP, MEGA1, MEGA2, ERY1, ERY2, ERY3. MEP: megakaryocyte-erythrocyte precursors (defined by CD34+ CD38+ IL-3Ra- CD45RA- ); MEGA1: megakaryocyte population 1 (defined by CD34+ CD61+ CD41+ CD45- ); MEGA2: megakaryocyte population 2 (defined by CD34- CD61+ CD41+ CD45- ); ERY1: erythrocyte population 1 (defined by CD34+ CD71+ GlyA- ); ERY2: erythrocyte population 2 (defined by CD34- CD71+ GlyA- ); ERY3: erythrocyte population 3 (defined by CD34- CD71+ GlyA+ ). Keywords: microRNA, miRNA, MEP, megakaryocyte, erythrocyte, lineage specification Varied numbers of samples were analyzed per population. Each sample came from one donor. Data were normalized as described (Lu et al., Nature 435, 834-838, 2005) with modifications. Average readings from water-only labeled samples were used for probe-specific background subtraction. Linear normalization among different bead sets for the same sample was performed using readings from 2 post-control probes with equal contribution. Sample normalization was subsequently carried out assuming equal total fluorescence readings.
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.
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.
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:Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT. To investigate possible mechanisms for its regulation of erythropoiesis we carried out RNAsequencing on Facs-sorted erythroblast population II (CD71+Ter119+) cells from Dhh-/-, Dhh+/- and WR mice.
Project description:Background: Recent advances in single-cell techniques have provided the opportunity to finely dissect cellular heterogeneity within populations previously defined by âbulkâ assays and to uncover rare cell types. In human hematopoiesis, megakaryocytes and erythroid cells differentiate from a shared precursor, the megakaryocyte-erythroid progenitor (MEP), which remains poorly defined.Results: To clarify the cellular pathway in erythro-megakaryocyte differentiation, we correlated the surface immunophenotype, transcriptional profile and differentiation potential of individual MEP cells. Highly purified, single MEP cells (n=681) were analyzed using index fluorescence-activated cell sorting with parallel targeted transcriptional profiling of the same cells performed using a specifically designed panel of 87 genes. Differentiation potential was tested in novel, single-cell differentiation assays. Our results demonstrated that immunophenotypic MEP in fact comprise three distinct subpopulations: (1) âPre-MEPâ, enriched for erythroid/megakaryocyte progenitors but with residual myeloid differentiation capacity (2) âE-MEPâ, strongly biased towards erythroid differentiation, and (3) âMK-MEPâ, a previously undescribed, rare population of cells that are bipotent but primarily generate megakaryocytic progeny. Therefore, conventionally-defined MEP are in fact a mixed population: a minority give rise to mixed-lineage colonies while the majority of cells are transcriptionally-primed to generate exclusively single-lineage output. Conclusions: Our study clarifies the cellular hierarchy in human megakaryocyte/erythroid lineage commitment and highlights the importance of using a combination of single-cell approaches to dissect cellular heterogeneity and identify rare cell types within a population. We present a novel immunophenotyping strategy that enables the prospective identification of specific intermediate progenitor populations in erythro-megakaryopoiesis, allowing for in-depth study of disorders including inherited cytopenias, myeloproliferative disorders and erythromegakaryocytic leukemias. Multiplex RT-PCR gene expression profiling of 807 human megakaryocyte-erythroid progenitor cells (MEP) isolated from three healthy donors by apheresis following G-CSF treatment. Cells were excluded if more than 70 assays did not result in amplification or displayed Ct higer than 13 for B2M or higher than 15 for GAPDH. Furthermore cells with a mean non-dropout Ct value greater than 20 were removed. This resulted in a dataset of 681 cells, which were subsequently normalised to the mean of B2M and GAPDH expression.
Project description:MEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells (HSPC) and in mature megakaryocytes. In contrast to its role in leukemogenesis, the role of MEIS1 in normal hematopoiesis is largely unknown. We show that MEIS1 can direct human hematopoietic progenitors towards a megakaryocyte-erythroid progenitor (MEP) fate. Ectopoic expression of MEIS1 in CD34+ cells resulted in increased erythroid differentiation at the expense of granulocyte and monocyte (GM) differentiation. MEIS1 overexpression not only skewed differentiation of CMPs towards the erythroid lineage but also reprogrammed GM progenitors towards erythrocyte differentiation. Expression profiling was used to determine the transcriptional changes induced by MEIS1 that lead to the oberved phenotype. A transcriptional program enriched for erythrocytic and megakaryocytic genes was detected.