Single Cell MK and HSC sequencing
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ABSTRACT:
PROVIDER: EGAS00001004844 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:Hematopoietic stem cells (HSCs) reside in the bone marrow (BM), can self-renew, and generate all blood cells. Most hematopoietic lineages arise through successive, increasingly lineage-committed progenitors. In contrast, megakaryocytes (MKs), hyperploid cells that generate platelets essential to hemostasis, can derive rapidly and directly from HSCs, subpopulations of which are primed for MK differentiation. The underlying mechanism is unknown however. Here we show that DNA damage and subsequent arrest in the G2 phase of the cell cycle rapidly induce MK commitment in HSCs, but not in progenitors, through an initially predominantly post-transcriptional mechanism. Cycling HSCs show extensive replication-induced DNA damage associated with uracil misincorporation in vivo and in vitro. Consistent with this notion, thymidine attenuated DNA damage, rescued HSC maintenance and reduced the generation of CD41+ MK-committed HSCs in vitro. Similarly, overexpression of the dUTP-scavenging enzyme, dUTPase, enhanced in vitro maintenance of HSCs. Replication stress-induced MK priming, at least in part caused by uracil misincorporation, is therefore a barrier to HSC maintenance and expansion. DNA damage-induced MK priming may allow rapid generation of a lineage essential to immediate organismal survival, while simultaneously removing damaged HSCs and potentially avoiding malignant transformation of self-renewing stem cells
2024-06-24 | GSE240695 | GEO
Project description:High ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of hematopoietic stem cells (HSC) and are responsible for platelet formation. Using a mouse knockout model with normal megakaryocyte numbers but essentially devoid of LCM (MK-LCM KO), we demonstrated a pronounced increase in bone marrow HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. When HSC isolated from a MK-LCM KO microenvironment were transplanted in lethally irradiated mice, the absence of LCM increased HSC in BM, blood and spleen. Severe thrombocytopenia was observed in animals with diminished LCM, although there was no change in megakaryocyte ploidy distribution. In contrast, WT HSC-generated LCM regulated a normal HSC pool and prevented thrombocytopenia. The present label-free quantitative LC-MSMS data was used to determine proteins that are differentially expressed in bone marrow cells of MK-LCM WT versus MK-LCM KO mice.
2023-05-10 | PXD040423 | Pride
Project description:The hematopoietic system gives rise to a heterogeneous population of terminally differentiated cells that reside in the bone marrow (BM) to fulfill their roles in immunity, blood clotting and tissue oxygenation. Hematopoietic stem and progenitor cells are at the apex of a hierarchically organized maturation cascade constantly replenishing the pool of differentiated cells to maintain blood cell homeostasis. The bone marrow microenvironment is functionally compartmentalized by heterogeneous niche cells that provide physical and soluble signals to spatio-temporally organize hematopoiesis. Megakaryocytes (MKs) are niche cells of hematopoietic origin that support hematopoietic stem cell (HSCs) homeostasis and generate circulating platelets. Platelet production involves the release of MK fragments while their cell body is entirely consumed in a process termed thrombopoiesis. Consequently, replenishment of fragmented MKs from MK progenitors (termed megakaryopoiesis) is required to ensure sufficient platelet production, but also to maintain MK homeostasis within the HSC niche. Here, we used intravital imaging of the megakaryocytic lineage to identify the spatio-temporal patterns of megakaryopoiesis during steady state and thrombocytopenia. We show that MK consumption during thrombopoiesis is compensated by immediate and local proliferation of MK progenitors. MK homeostasis is controlled by plasmacytoid dendritic cells (pDCs) that scan the BM and secrete Interferon alpha (INFa) upon detection of exhausted megakaryocytes to trigger megakaryopoiesis. We identified local pDC-derived INFa release as a physiological inflammatory stimulus of the bone marrow niche that synchronizes megakaryopoiesis and thrombopoiesis to maintain homeostasis of MKs and platelets in steady state and under stress. Viral infection with SARS-CoV-2 can manipulate pDC-driven MK proliferation leading to inappropriate megakaryopoiesis, which has been associated with thrombotic complications during COVID-19.
2024-04-26 | GSE185488 | GEO
Project description:During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine Thrombopoietin (Thpo) concomitantly regulates platelet production and maintains HSC stem cell potential whether Thpo directly controls Mk-lineage differentiation of HSCs is unclear. Stress hematopoiesis requires quiescent HSCs to proliferate, a process which depends on a higher energy production. However, whether this switch of metabolic state relates to lineage differentiation of HSCs is not known. We here show that Thpo rapidly upregulates mitochondrial activity in HSCs which was accompanied by preferential differentiation to Mk-lineage. During unperturbed hematopoiesis, HSCs with high mitochondrial content and activity exhibit Mk-lineage biased differentiation. Furthermore, Thpo rapidly skewed HSCs to express a tetraspanin molecule, CD9, which expression correlated to mitochondria cell content. While highly proliferative, mitochondrial-rich HSCs were resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo regulated mitochondrial activity did not associate with high levels of cMpl expression but was influenced by non-nuclear mitochondrial translocation of phosphorylated of STAT3 at serine 727. Our data reveals that HSCs are metabolically heterogeneous and higher mitochondrial activity primes HSCs toward the Mk lineage differentiation. We also uncover a pivotal role of Thpo in regulating the rapid Mk-lineage commitment during stress hematopoiesis. Our findings suggest that mitochondria metabolism primes HSCs not only to exit dormancy but toward direct differentiation to Mk lineage.
2018-12-31 | GSE121001 | GEO
Project description:The mechanism underlying thrombocytosis in patients with iron deficiency anemia remains unknown. We present findings that support the hypothesis that low iron biases the commitment of Megakaryocytic-Erythroid Progenitors (MEP) toward the megakaryocytic (Mk) lineage in mice. In MEP of Transmembrane serine protease 6 knockout (Tmprss6-/-) mice, which exhibit iron deficiency microcytic anemia concomitant with thrombocytosis, we observed a megakaryocytic (Mk) bias, decreased labile iron, and decreased proliferation relative to wild-type (WT) MEP. Bone marrow transplantation assays suggest that the systemic iron deficiency of the Tmprss6-/- recipients contributes to the MEP lineage commitment bias. Genes involved in metabolic, VEGF, and ERK pathways were enriched among those differentially expressed between WT and Tmprss6-/- MEP. Corroborating our findings from the murine model of chronic iron deficiency anemia, primary human MEP also exhibited decreased proliferation and Mk-biased commitment when their iron sensing pathways were disrupted by knockdown of Transferrin Receptor 2. These data are consistent with a model in which low iron in the marrow environment affects MEP metabolism, attenuates ERK signaling, slows proliferation, and biases MEP toward Mk lineage commitment. Keywords: megakaryocytic-erythroid progenitor (MEP), iron deficiency, Tmprss6
2023-03-17 | GSE227563 | GEO
Project description:An increased number of circulating CD34+ hematopoietic progenitors (HP) and a prominent amplification of dystrophic megakaryocytes (MK) are observed in PMF patients. As transcriptome data from CD34+ hematopoietic progenitors showed modulations of FLT3 and MAP kinase expression independently of the JAK2V617F mutation status Transcriptome analysis was performed on circulating CD34+ cells from PMF patients using Agilent 22K microarray and compared to CD34+ cells from blood and bone marrow from un-mobilized healthy donors. Indirect map: each tested sample was hybridzed with reference probe
2008-07-29 | E-GEOD-12234 | biostudies-arrayexpress
Project description:During adult bone marrow hematopoiesis, extremely rare and dormant hematopoietic stem cells (HSCs) harbor the highest self-renewal activity within all blood cells. They give rise to active HSCs, which generate multipotent progenitors (MPPs) which differentiate into lineage-committed progenitors and subsequently mature cells. While HSCs are characterized by long-term self-renewal capacity, quiescence and multipotency, MPPs show steadily decreasing self-renewal activity, are cycling but are thought to maintain multipotency. To establish a comprehensive genome-wide landscape of expressed transcripts, we performed a quantitative transcriptome analysis by next-generation sequencing (RNA-seq) of seven ex vivo FACS-sorted mouse HSC/progenitor populations. Eleven-fold coverage of the genome was achieved, revealing quantification of > 27,000 mRNA species of which 589 long non-coding RNAs (lncRNAs) were quantified. A profile of 79 differentially expressed lncRNAs in HSC-MPPs was identified suggesting a role for these RNA species in HSC/progenitor biology. Expression clusters of transcription factors and cell adhesion molecules are identified between the different cell populations. Dormant HSCs, as identified by label-retaining assays, showed a highly differential expression profile compared to active HSCs. In addition to >200 differentially expressed cell surface receptors and lncRNAs, processes including metabolism, development, immune response, signaling (TGFb, Kit, senescence/autophagy) are distinct between the two types of HSCs. In addition, using whole cell proteome analysis of FACS-sorted HSCs and MPP1, >6,000 proteins were identified by quantitative tandem mass spectrometry. Quantification of these proteins confirmed the close relationship between these cell types also seen in their transcript profile and revealed processes such as energy metabolism, immune response and cell cycle to be modulated along early lineage progression. While MPP1/2 still show multilineage potential in reconstitution experiments, a strong lineage bias and low self-renewal potential is observed in mice reconstituted with MPP3/4. These functional differences are accompanied by complex changes in their transcriptome and is also revealed by principal component analysis. In summary, the global mRNA, lncRNA and proteome signatures uncovered here and which are complemented by functional assays, provide a comprehensive and searchable resource of the molecular make-up of the entire HSC/progenitor population present in the bone marrow. These data will provide the basis for a global understanding of stem cell biology in the adult blood system. The uploaded dataset corresponds to the quantitative proteomic comparison of HSC and MPP1, which was done in three biological replicates. Data analysis: MS raw data files were processed with MaxQuant (version 1.3.0.5) (Cox and Mann 2008). Enzyme specificity was set to trypsin/P and a maximum of two missed cleavages were allowed. Cysteine carbamidomethylation and methionine oxidation were selected as fixed and variable modifications, respectively. The derived peak list was searched using the built-in Andromeda search engine (version 1.3.0.5) in MaxQuant against the Uniprot mouse database (2013.02.20) containing 75,721 proteins to which 247 frequently observed contaminants as well as reversed sequences of all entries had been added. Initial maximal allowed mass tolerance was set to 20 ppm for peptide masses, followed by 6 ppm in the main search, and 0.5 Dalton for fragment ion masses. The minimum peptide length was set to six amino acid residues and three labeled amino acid residues were allowed. A 1% false discovery rate (FDR) was required at both the protein level and the peptide level. In addition to the FDR threshold, proteins were considered identified if they had at least one unique peptide. The protein identification was reported as an indistinguishable “protein group” if no unique peptide sequence to a single database entry was identified. The ‘match between runs’ was enabled for consecutive peptide fractions with a 2 minutes time window. The iBAQ algorithm was used for estimation of the abundance of different proteins within a single sample (proteome) (Schwanhausser 2011). For evaluation of differential protein expression between HSC and MPP1, statistical analysis was performed for the proteins quantified in all three replicates using the Limma package in R/Bioconductor (Gentleman 2004, Smyth 2004). After fitting a linear model to the data, an empirical Bayes moderated t-test was used for the protein ratios, which were weighted on log10(summed peptide intensities) in order to capture the effect that the statistical spread of unregulated proteins is much more focused for highly abundant proteins than for low abundance ones (Cox 2008). P-values were then adjusted for multiple testing with Benjamini and Hochberg's method and proteins with an adjusted p-value lower than 0.1 were considered to be differentially expressed between HSC and MPP1. Associated transcriptomics data has been deposited at ArrayExpress with accession E-MTAB-2262.
2014-08-21 | PXD000572 | Pride
Project description:This study aimed to compare the expression profile of GzmB deficient HSCs and their WT counterparts in order to understand their phenotype in bone marrow in vivo reconstitution assays.
2014-04-11 | GSE56574 | GEO
Project description:RNA-seq expression analysis of transcripts encoding proteasome subunits in human CD34+ cord blood cell-derived megakaryocytes and mouse bone marrow-derived megakaryocytes. Analysis of transcript expression in human CD34+ cord blood cell-derived megakaryocytes and mouse bone marrow-derived megakaryocytes.
2015-01-31 | E-GEOD-58202 | biostudies-arrayexpress
Project description:This study describes the changes in epigenetic chromatin modifications during murine hematopoietic stem cell differentiation in vivo using a modified miniChIP-chip technology. We have addressed issues including bivalent (H3K4me3/H3K27me3) modifications, lineage priming hypothesis, and stem cell chromatin properties in our study described in Weishaupt et al., 2009 (Blood) Comparison of 4 murine hematopoietic stem, progenitor and mature cell types directly isolated from primary tissues using FACS. HSCs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150+, Flk2/Flt3- (LSKCD150+ cells). MPPs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150-, Flk2/Flt3+ (LSKCD150- cells). PreMegEs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1-, CD150+, CD105-, FcgRI/IIlo and CD41- cells as described in Pronk et al., 2007. Splenic-derived CD4+ T cells are phentypically identified as CD4+, CD8-, B220-, Nk1.1- cells as described in Rolf et al., 2008.
2009-11-08 | E-GEOD-18734 | biostudies-arrayexpress