Single Cell Transcriptomic Analysis of Lung and Hematopoietic Megakaryocytes from Embryonic and Adult Mice.
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE152574 | GEO | 2021/01/04
REPOSITORIES: GEO
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Project description:Srf is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. In order to test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F (KO) mice are born with normal mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased numbers and percentages of CD41+ megakaryocytes (WT: 0.41+/-0.06%; KO: 1.92+/-0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by both FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are downregulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production, due at least in part, to regulation of cytoskeletal genes. C-kit+CD41+ megakaryocyte progenitors from PF4-Cre/SRF C57BL/6 SRF WT (3) and C57BL/6 SRF KO (3) mice were sorted by flow cytometry and cultured for three days in thrombopoietin.
2010-07-13 | E-GEOD-21859 | biostudies-arrayexpress
Project description:To clarigy the molecular mechanism underlying the IMiD-induced megakaryocytic lineage commitment in human megakaryocyte/erythrocyte progenitors, we performed transcriptome analysis of CD41-CD105+ and CD41+CD105- cells obtained from megakaryocyte/erythrocyte progenitors treated with lenalidomide for 72 hours. Differentially expressed genes were comprehensively evaluated by gene set enrichment analysis. CD105-CD41+ cells showed the enrichment in the expression of genes related to megakaryocytes and platelet, whereas genes related to erythroid lineage were negatively enriched. CD41+CD105- cells expressed higher levels of transcription factors required for megakaryocyte differentiation including FLI1, RUNX1, and MEIS1. Furthermore, we performed a knockdown strategy with small interfering RNA against MEIS1 mRNA, confirming that lenalidomide-induced megakaryocyte commitment should be due to the up-regulation of MEIS1 in megakaryocyte/erythrocyte progenitors.
2021-11-25 | GSE162212 | GEO
Project description:Srf is a MADS-box transcription factor that is critical for muscle differentiation. Its function in hematopoiesis has not yet been revealed. Mkl1, a cofactor of Srf, is part of the t(1;22) translocation in acute megakaryoblastic leukemia, and plays a critical role in megakaryopoiesis. In order to test the role of Srf in megakaryocyte development, we crossed Pf4-Cre mice, which express Cre recombinase in cells committed to the megakaryocytic lineage, to SrfF/F mice in which functional Srf is no longer expressed after Cre-mediated excision. Pf4-Cre/SrfF/F (KO) mice are born with normal mendelian frequency, but have significant macrothrombocytopenia with approximately 50% reduction in platelet count. In contrast, the BM has increased numbers and percentages of CD41+ megakaryocytes (WT: 0.41+/-0.06%; KO: 1.92+/-0.12%) with significantly reduced ploidy. KO mice show significantly increased megakaryocyte progenitors in the BM by both FACS analysis and CFU-Mk. Megakaryocytes lacking Srf have abnormal stress fiber and demarcation membrane formation and platelets lacking Srf have abnormal actin distribution. In vitro and in vivo assays reveal platelet function defects in KO mice. Critical actin cytoskeletal genes are downregulated in KO megakaryocytes. Thus, Srf is required for normal megakaryocyte maturation and platelet production, due at least in part, to regulation of cytoskeletal genes.
2010-07-13 | GSE21859 | GEO
Project description:Background: Children with Down syndrome (DS) are predisposed to acute megakaryoblastic leukemia (AMKL, or AML M7) and a related myeloid disorder, referred to as transient myeloproliferative disorder (TMD), or transient leukemia (TL). Recently, acquired mutations in the erythroid/megakaryocytic lineage-specific transcription factor GATA-1 have been identified in megakaryoblasts from virtually all DS patients with AMKL (DS-AMKL), as well as in nearly all DS neonates with TMD. These mutations prevent synthesis of full-length GATA-1, but lead to synthesis of a truncated GATA-1 protein (GATA-1s) that lacks the N-terminal transactivation domain. To determine the in vivo requirement of GATA-1 N-terminal domain in hematopoiesis and to model DS megakaryocytic leukemia initiated by GATA-1 mutation, we generated a GATA-1 knockin allele with a truncation at this domain (GATA-1deltaN). ES cells bearing this mutant allele generated a unique type of large Thrombopoietin (Tpo)-dependent megakaryocytes-containing colonies, when differentiated in vitro. Fetal livers from mice carrying this mutant allele contained elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. In in vitro hematopoietic colony assays, mutant cells from yolk sacs and mid-gestation fetal livers gave rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). Consistent with that, when mutant fetal liver progenitors were cultured in liquid medium in the presence of Tpo, the derived megakaryocytes displayed marked hyperproliferation and grew for longer period of time than WTs. Specific aims: To understand the molecular basis for the megakaryocytic hyperproliferation phenotype of the GATA-1deltaN mutants, we plan to profile gene expression patterns of fetal liver-derived primary megakaryocytes and megakaryocytic progenitors from the GATA-1deltaN mutants and compare them with WTs, as well as GATA-1deltaNeodeltaHS mutants (GATA-1– megakaryocytes). Experimental design: E12.5 fetal livers were harvested from either GATA-1 WT and mutant embryos. Single cell suspensions were made from these livers and the cells were cultured in liquid medium in the presence of Tpo for 3 days. On day 3, primary megakaryocytes were enriched by a BSA gradient, and further enriched by anti-CD41 antibody and magnetic microbeads. Megakaryocytic progenitors were collected by FACS sorting for small cells in the same culture that express low level of CD41. Total RNAs were then prepared from these two populations and submitted for microarray profiling. Conclusions: When examining genes that are normally upregulated upon megakaryocyte differentiation (many of this class are megakaryocyte-specific genes), we found in GATA-1deltaN megakaryocytes, the majority of these genes are upregulated, although not to the full extent as seen in WTs. In contrast, this class of genes is largely not upregulated in GATA-1deltaNeodeltaHS megakaryocytes, which are blocked at an immature stage of differentiation. Genes that are downregulated during megakaryocyte differentiation include those that are normally repressed by GATA-1. Among genes that are not properly downregulated in GATA-1deltaN megakaryocytes, five transcription factors (Myc, Myb, GATA-2, PU.1, Ikaros) are of particular interest. Inadequate GATA-1s-mediated repression of transcription factors required for proliferation of hematopoietic progenitors (Myc, Myb and GATA-2) and for specifying lineage choices other than megakaryocyte/erythrocyte (PU.1 and Ikaros) may contribute to hyperproliferation of GATA-1deltaN megakaryocytes. Keywords = GATA-1 Keywords = megakaryocyte Keywords = mouse model Keywords: other
2005-05-14 | GSE2433 | GEO
Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells. Gene expression was measured in primary megakaryocytes cultured from p45-/- fetal livers and wild type fetal livers at E13.5. Three independent experiments were performed.
2013-08-07 | E-GEOD-35645 | biostudies-arrayexpress
Project description:Background: Children with Down syndrome (DS) are predisposed to acute megakaryoblastic leukemia (AMKL, or AML M7) and a related myeloid disorder, referred to as transient myeloproliferative disorder (TMD), or transient leukemia (TL). Recently, acquired mutations in the erythroid/megakaryocytic lineage-specific transcription factor GATA-1 have been identified in megakaryoblasts from virtually all DS patients with AMKL (DS-AMKL), as well as in nearly all DS neonates with TMD. These mutations prevent synthesis of full-length GATA-1, but lead to synthesis of a truncated GATA-1 protein (GATA-1s) that lacks the N-terminal transactivation domain. To determine the in vivo requirement of GATA-1 N-terminal domain in hematopoiesis and to model DS megakaryocytic leukemia initiated by GATA-1 mutation, we generated a GATA-1 knockin allele with a truncation at this domain (GATA-1deltaN). ES cells bearing this mutant allele generated a unique type of large Thrombopoietin (Tpo)-dependent megakaryocytes-containing colonies, when differentiated in vitro. Fetal livers from mice carrying this mutant allele contained elevated numbers of CD41+ cells throughout embryonic development, especially during mid-gestation. In in vitro hematopoietic colony assays, mutant cells from yolk sacs and mid-gestation fetal livers gave rise to a large number of macroscopic hyperproliferative megakaryocytic colonies (CFU-MKs). Consistent with that, when mutant fetal liver progenitors were cultured in liquid medium in the presence of Tpo, the derived megakaryocytes displayed marked hyperproliferation and grew for longer period of time than WTs. Specific aims: To understand the molecular basis for the megakaryocytic hyperproliferation phenotype of the GATA-1deltaN mutants, we plan to profile gene expression patterns of fetal liver-derived primary megakaryocytes and megakaryocytic progenitors from the GATA-1deltaN mutants and compare them with WTs, as well as GATA-1deltaNeodeltaHS mutants (GATA-1megakaryocytes). Experimental design: E12.5 fetal livers were harvested from either GATA-1 WT and mutant embryos. Single cell suspensions were made from these livers and the cells were cultured in liquid medium in the presence of Tpo for 3 days. On day 3, primary megakaryocytes were enriched by a BSA gradient, and further enriched by anti-CD41 antibody and magnetic microbeads. Megakaryocytic progenitors were collected by FACS sorting for small cells in the same culture that express low level of CD41. Total RNAs were then prepared from these two populations and submitted for microarray profiling. Conclusions: When examining genes that are normally upregulated upon megakaryocyte differentiation (many of this class are megakaryocyte-specific genes), we found in GATA-1deltaN megakaryocytes, the majority of these genes are upregulated, although not to the full extent as seen in WTs. In contrast, this class of genes is largely not upregulated in GATA-1deltaNeodeltaHS megakaryocytes, which are blocked at an immature stage of differentiation. Genes that are downregulated during megakaryocyte differentiation include those that are normally repressed by GATA-1. Among genes that are not properly downregulated in GATA-1deltaN megakaryocytes, five transcription factors (Myc, Myb, GATA-2, PU.1, Ikaros) are of particular interest. Inadequate GATA-1s-mediated repression of transcription factors required for proliferation of hematopoietic progenitors (Myc, Myb and GATA-2) and for specifying lineage choices other than megakaryocyte/erythrocyte (PU.1 and Ikaros) may contribute to hyperproliferation of GATA-1deltaN megakaryocytes.
2007-08-31 | E-GEOD-2433 | biostudies-arrayexpress
Project description:Dominant-negative mutations in transcription factor Growth Factor Independence-1B (GFI1B) cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that normal and mutant GFI1B interacted most strongly with the LSD1-RCOR-HDAC corepressor complex in megakaryoblasts. Sequestration of this complex by mutant GFI1B and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1B-mutant induced pluripotent stem cells (iPSC) also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant iPSC-derived megakaryocytes identified a multitude of deregulated pathways downstream of mutant GFI1B. Proteome studies on primary normal and GFI1B-mutant platelets showed reduced expression of proteins implicated in platelet function, and sustained expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B regulates a broad developmental program during megakaryopoiesis. Mutant GFI1B deregulates this program through LSD1-RCOR-HDAC sequestering.
2019-01-25 | PXD009020 | Pride
Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells.
2013-08-07 | GSE35645 | GEO
Project description:Ex vivo differentiation of megakaryocytes (Mk) was carried out using human cord blood (CB) CD34+ cells under the stimulation of recombinant human interleukin-3, stem cell factor, and thrombopoietin for 7 days, followed by thrombopoietin treatment alone for further 3 days. Total cellular RNA was extracted from Day-0 CD34+, Day-10 CD41+ and CD41- cells, respectively. Microarray was performed and the data was analyzed using the GeneChip Operating Software, Spotfire DecisionSite Software and Genomatix Application Software.
2009-03-25 | E-MEXP-1546 | biostudies-arrayexpress
Project description:We developed a novel differentiation system that directionally induces hESCs into megakaryocytes and functional platelet in vitro by highly mimicking the in vivo developmental process of megakaryocytes and platelets. We then performed gene expression profiling analysis using data obtained by RNA-seq at different stages of differentiation during the differentiation of hESCs into megakaryocyte lineages.
2024-08-14 | GSE216593 | GEO