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Cell Stem Cell Mar 2021Despite our growing understanding of embryonic immune development, rare early megakaryocytes (MKs) remain relatively understudied. Here we used single-cell RNA...
Despite our growing understanding of embryonic immune development, rare early megakaryocytes (MKs) remain relatively understudied. Here we used single-cell RNA sequencing of human MKs from embryonic yolk sac (YS) and fetal liver (FL) to characterize the transcriptome, cellular heterogeneity, and developmental trajectories of early megakaryopoiesis. In the YS and FL, we found heterogeneous MK subpopulations with distinct developmental routes and patterns of gene expression that could reflect early functional specialization. Intriguingly, we identified a subpopulation of CD42bCD14 MKs in vivo that exhibit high expression of genes associated with immune responses and can also be derived from human embryonic stem cells (hESCs) in vitro. Furthermore, we identified THBS1 as an early marker for MK-biased embryonic endothelial cells. Overall, we provide important insights and invaluable resources for dissection of the molecular and cellular programs underlying early human megakaryopoiesis.
Topics: Cell Differentiation; Endothelial Cells; Human Embryonic Stem Cells; Humans; Megakaryocytes; Thrombopoiesis
PubMed: 33340451
DOI: 10.1016/j.stem.2020.11.006 -
Immunity Dec 2022Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that...
Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.
Topics: Humans; Megakaryocytes; Thrombopoiesis; Neutrophils; Cardiovascular Diseases; Blood Platelets; Thrombosis; Myocardial Infarction
PubMed: 36272416
DOI: 10.1016/j.immuni.2022.10.001 -
Arteriosclerosis, Thrombosis, and... Jul 2019Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular... (Review)
Review
Megakaryocytes are hematopoietic cells, which are responsible for the production of blood platelets. The traditional view of megakaryopoiesis describes the cellular journey from hematopoietic stem cells, through a hierarchical series of progenitor cells, ultimately to a mature megakaryocyte. Once mature, the megakaryocyte then undergoes a terminal maturation process involving multiple rounds of endomitosis and cytoplasmic restructuring to allow platelet formation. However, recent studies have begun to redefine this hierarchy and shed new light on alternative routes by which hematopoietic stem cells are differentiated into megakaryocytes. In particular, the origin of megakaryocytes, including the existence and hierarchy of megakaryocyte progenitors, has been redefined, as new studies are suggesting that hematopoietic stem cells originate as megakaryocyte-primed and can bypass traditional lineage checkpoints. Overall, it is becoming evident that megakaryopoiesis does not only occur as a stepwise process, but is dynamic and adaptive to biological needs. In this review, we will reexamine the canonical dogmas of megakaryopoiesis and provide an updated framework for interpreting the roles of traditional pathways in the context of new megakaryocyte biology. Visual Overview- An online visual overview is available for this article.
Topics: Bone Marrow Cells; Cell Communication; Cell Cycle; Cell Differentiation; Cell Lineage; Hematopoietic Stem Cells; Humans; Inflammation; Megakaryocytes; Signal Transduction; Thrombopoiesis; Thrombopoietin; Transcription Factors
PubMed: 31043076
DOI: 10.1161/ATVBAHA.119.312129 -
Cell Sep 2023Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å...
Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPO) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPO decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPO in hematology research and clinical HSC transplantation.
Topics: Animals; Humans; Mice; Cell Cycle; Cryoelectron Microscopy; Receptors, Thrombopoietin; Thrombopoiesis; Thrombopoietin; DNA Methylation
PubMed: 37633268
DOI: 10.1016/j.cell.2023.07.037 -
Blood May 2022Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains...
Translation is essential for megakaryocyte (MK) maturation and platelet production. However, how the translational pathways are regulated in this process remains unknown. In this study, we found that MK/platelet-specific lactate dehydrogenase A (LdhA) knockout mice exhibited an increased number of platelets with remarkably accelerated MK maturation and proplatelet formation. Interestingly, the role of LDHA in MK maturation and platelet formation did not depend on lactate content, which was the major product of LDHA. Mechanism studies revealed that LDHA interacted with eukaryotic elongation factor 2 (eEF2) in the cytoplasm, controlling the participation of eEF2 in translation at the ribosome. Furthermore, the interaction of LDHA and eEF2 was dependent on nicotinamide adenine dinucleotide (NADH), a coenzyme of LDHA. NADH-competitive inhibitors of LDHA could release eEF2 from the LDHA pool, upregulate translation, and enhance MK maturation in vitro. Among LDHA inhibitors, stiripentol significantly promoted the production of platelets in vivo under a physiological state and in the immune thrombocytopenia model. Moreover, stiripentol could promote platelet production from human cord blood mononuclear cell-derived MKs and also have a superposed effect with romiplostim. In short, this study shows a novel nonclassical function of LDHA in translation that may serve as a potential target for thrombocytopenia therapy.
Topics: Animals; Blood Platelets; Elongation Factor 2 Kinase; Enzyme Inhibitors; L-Lactate Dehydrogenase; Megakaryocytes; Mice; Mice, Knockout; NAD; Peptide Elongation Factor 2; Thrombocytopenia; Thrombopoiesis
PubMed: 35176139
DOI: 10.1182/blood.2022015620 -
Signal Transduction and Targeted Therapy Oct 2022Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the...
Immune thrombocytopenia (ITP) is an autoimmune disorder, in which megakaryocyte dysfunction caused by an autoimmune reaction can lead to thrombocytopenia, although the underlying mechanisms remain unclear. Here, we performed single-cell transcriptome profiling of bone marrow CD34 hematopoietic stem and progenitor cells (HSPCs) to determine defects in megakaryopoiesis in ITP. Gene expression, cell-cell interactions, and transcriptional regulatory networks varied in HSPCs of ITP, particularly in immune cell progenitors. Differentially expressed gene (DEG) analysis indicated that there was an impaired megakaryopoiesis of ITP. Flow cytometry confirmed that the number of CD9 and HES1 cells from LinCD34CD45RA HSPCs decreased in ITP. Liquid culture assays demonstrated that CD9LinCD34CD45RA HSPCs tended to differentiate into megakaryocytes; however, this tendency was not observed in ITP patients and more erythrocytes were produced. The percentage of megakaryocytes differentiated from CD9LinCD34CD45RA HSPCs was 3-fold higher than that of the CD9 counterparts from healthy controls (HCs), whereas, in ITP patients, the percentage decreased to only 1/4th of that in the HCs and was comparable to that from the CD9 HSPCs. Additionally, when co-cultured with pre-B cells from ITP patients, the differentiation of CD9LinCD34CD45RA HSPCs toward the megakaryopoietic lineage was impaired. Further analysis revealed that megakaryocytic progenitors (MkP) can be divided into seven subclusters with different gene expression patterns and functions. The ITP-associated DEGs were MkP subtype-specific, with most DEGs concentrated in the subcluster possessing dual functions of immunomodulation and platelet generation. This study comprehensively dissects defective hematopoiesis and provides novel insights regarding the pathogenesis of ITP.
Topics: Bone Marrow; Humans; Purpura, Thrombocytopenic, Idiopathic; Thrombocytopenia; Thrombopoiesis; Transcriptome
PubMed: 36202780
DOI: 10.1038/s41392-022-01167-9 -
Cell Jul 2018The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing...
The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.
Topics: Bioreactors; Blood Platelets; Cell Culture Techniques; Humans; Hydrodynamics; Induced Pluripotent Stem Cells; Megakaryocytes; Thrombopoiesis
PubMed: 30017246
DOI: 10.1016/j.cell.2018.06.011 -
Signal Transduction and Targeted Therapy Jun 2021Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in...
Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-β released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.
Topics: Adolescent; Adult; Animals; Female; Humans; Macrophages; Male; Mice; Mice, Knockout; Middle Aged; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Thrombocytopenia; Thrombopoiesis
PubMed: 34140465
DOI: 10.1038/s41392-021-00627-y -
Experimental Hematology Jan 2018In mammals, platelets are produced in the blood by cytoplasmic fragmentation of megakaryocytes (MKs). Platelet production is thus dependent on both the MK number and... (Review)
Review
In mammals, platelets are produced in the blood by cytoplasmic fragmentation of megakaryocytes (MKs). Platelet production is thus dependent on both the MK number and size. During differentiation, MKs switch from a division by mitosis to polyploidization by endomitosis to increase their size. The endomitotic process includes several successive rounds of DNA replication with an entry in mitosis with a failure in late cytokinesis and a defect in karyokinesis. This leads to a giant cell with a modal ploidy at 16N and one multilobulated nucleus. The entire genome is duplicated several times and all alleles remain functional producing a hypermetabolic cell. A defect in abscission explains the cytokinesis failure and is related to an altered accumulation of actomyosin at the cleavage furrow as a consequence of both a low local RhoA activity and silencing of the MYH10 gene. This mechanism is regulated by transcription factors that govern differentiation explaining the intricacies of both processes. However, the endomitotic cell cycle regulation is still incompletely understood, particularly mitosis entry, escape to the tetraploid checkpoint, and defect in karyokinesis. Polyploidization is regulated during ontogeny, the first embryonic MKs being 2N. The molecular mechanism of this embryo-fetal/adult transition is beginning to be understood. In physiological conditions, MK ploidy is increased by an enhanced platelet demand through the thrombopoietin/myeloproliferative leukemia axis. In numerous hematologic malignancies, MK ploidy decreases, but it is always associated with a defect in MK differentiation. It has been proposed that polyploidization induction could be a treatment for some malignant MK disorders.
Topics: Animals; Cell Cycle Checkpoints; Cell Cycle Proteins; Cytokinesis; DNA Replication; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Hematologic Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Mammals; Megakaryocytes; Mitosis; Molecular Targeted Therapy; Myelodysplastic Syndromes; Polyploidy; Signal Transduction; Thrombopoiesis; Thrombopoietin; Transcription Factors; rhoA GTP-Binding Protein
PubMed: 29111429
DOI: 10.1016/j.exphem.2017.10.001 -
Nature Communications Apr 2023Thrombocytopenia is a major complication in a subset of patients with multiple myeloma (MM). However, little is known about its development and significance during MM....
Thrombocytopenia is a major complication in a subset of patients with multiple myeloma (MM). However, little is known about its development and significance during MM. Here, we show thrombocytopenia is linked to poor prognosis in MM. In addition, we identify serine, which is released from MM cells into the bone marrow microenvironment, as a key metabolic factor that suppresses megakaryopoiesis and thrombopoiesis. The impact of excessive serine on thrombocytopenia is mainly mediated through the suppression of megakaryocyte (MK) differentiation. Extrinsic serine is transported into MKs through SLC38A1 and downregulates SVIL via SAM-mediated tri-methylation of H3K9, ultimately leading to the impairment of megakaryopoiesis. Inhibition of serine utilization or treatment with TPO enhances megakaryopoiesis and thrombopoiesis and suppresses MM progression. Together, we identify serine as a key metabolic regulator of thrombocytopenia, unveil molecular mechanisms governing MM progression, and provide potential therapeutic strategies for treating MM patients by targeting thrombocytopenia.
Topics: Humans; Bone Marrow; Thrombopoiesis; Multiple Myeloma; Thrombocytopenia; Bone Marrow Cells; Megakaryocytes; Blood Platelets; Tumor Microenvironment
PubMed: 37055385
DOI: 10.1038/s41467-023-37699-z