Project description:Integration of index sorting and single cell functional assays identified two functionally distinct subsets of phenotypic haematopoietic stem cells and multipotent progenitors (HSC/MPPs) in the peripheral blood (PB) from healthy individuals. CD71- HSC/MPPs from PB are multipotent and can repopulate the NSG xenograft model. CD71+ HSC/MPPs are a subset of phenotypic HSC/MPPs that is uniquely restricted to give rise to erythroid and megakaryocytic lineages, and expands in conditions with chronic stimulation of platelet production (e.g. frequent platelet donation, idiopathic thrombocytopenic purpura, essential thrombocythaemia). Here, we report the bulk transcriptomes of pools of 20 cells from CD71- HSC/MPPs (CD19- CD38- CD45RA- CD34lo CD71-) and CD71+ HSC/MPPs (CD19- CD38- CD45RA- CD34lo CD71+). Altogether the data shows the transcriptional similarities and differences between both subsets. It shows, that the unique erythroid/megakaryocytic lineage-priming of CD71+ HSC/MPPs is already initiated at transcriptional level, and that CD71+ HSC/MPPs differ from CD71- HSC/MPPs with regards to their protein synthesis/metabolic pathways.
Project description:Knockdown of HSPA9 causes a dose-dependent decrease in erythroid maturation of CD34+ cells differentiated in culture. Due to differences in the degree of differentiation, a more homogeneous population was selected for using FACS and the gene expression profile of these cells was compared. We used a lentiviral vector (pLKO.1) expressing short hairpin RNAs targeting either luciferase (control shLUC) or HSPA9 (shHSPA9-433) to knock down expression of HSPA9. We isolated CD34+ cells from human cord blood (Day 0), transduced cells with a lentiviral vector (Day 1), selected for transduced cells with puromycin and differentiated them in erythroid culture media before FACS isolation of the CD34+/CD71- population (Day 5). Four independent CD34+ populations were isolated, differentiated and sorted for biologic replicates.
Project description:We report that Nup358, a nucleoporin linked to acute myeloid leukemia and myeloproliferative neoplasms, is required for the developmental progression of early myeloid progenitors. We found that loss of Nup358 in mice is associated with the accumulation of myeloid-primed multipotent progenitors (MPPs) in bone marrow and the loss of myeloid-committed progenitors and mature myeloid cells. Accumulated MPPs in Nup358 knockout mice are greatly restricted to megakaryocyte/erythrocyte biased MPP2 cells and fail to progress into committed myeloid progenitors.
Project description:Transcription cofactor Rcor1 has been linked biochemically both to neurogenesis and hematopoiesis. Here we studied the function of Rcor1 in vivo and showed it is essential to erythropoeisis during embryonic development. Rcor1 mutant proerythroblasts, unlike normal cells, can form myeloid colonies in vitro. To investigate the underlying molecular mechanisms for block of erythropoiesis and increased myeloid potential, we used RNA-seq to reveal the differentially expressed genes from erythroid progenitors due to depletion of Rcor1. RNA were extracted from FACS sorted CD71+,TER119- erythroid progenitors from control (Rcor1+/+ and Rcor1+/-) or Mutant (Rcor1-/- ) E13.5 fetal liver. Each library was made by pooling RNA from several fetal livers. Two biological replicates were made for either control or mutant condition.
Project description:CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3 CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3
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.
