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Scientific Reports Dec 2023The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from...
The use of megakaryoblastic leukemia MEG-01 cells can help reveal the mechanisms of thrombopoiesis. However, conventional in vitro activation of platelet release from MEG-01 cells requires thrombopoietin, which is costly. Here, we aim to develop a more straightforward and affordable method. Synchronization of the MEG-01 cells was initially performed using serum-free culture, followed by spontaneous cell differentiation in the presence of serum. Different stages of megakaryoblast differentiation were classified based on cell morphology, DNA content, and cell cycle. The MEG-01 cells released platelet-like particles at a level comparable to that of the thrombopoietin-activated MEG-01 cells. The platelet-like particles were distinguishable from PLP-derived extracellular vesicles and could express P-selectin following ADP activation. Importantly, the platelet-like particles induced fibrin clotting in vitro using platelet-poor plasma. Therefore, this thrombopoietin-independent cell synchronization method is an effective and straightforward method for studying megakaryopoiesis and thrombopoiesis.
Topics: Megakaryocytes; Thrombopoietin; Megakaryocyte Progenitor Cells; Blood Platelets; Thrombopoiesis
PubMed: 38110522
DOI: 10.1038/s41598-023-50111-6 -
Critical shifts in lipid metabolism promote megakaryocyte differentiation and proplatelet formation.Nature Cardiovascular Research Sep 2023During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a...
During megakaryopoiesis, megakaryocytes (MK) undergo cellular morphological changes with strong modification of membrane composition and lipid signaling. Here we adopt a lipid-centric multiomics approach to create a quantitative map of the MK lipidome during maturation and proplatelet formation. Data reveal that MK differentiation is driven by an increased fatty acyl import and lipid synthesis, resulting in an anionic membrane phenotype. Pharmacological perturbation of fatty acid import and phospholipid synthesis blocked membrane remodeling and directly reduced MK polyploidization and proplatelet formation resulting in thrombocytopenia. The anionic lipid shift during megakaryopoiesis was paralleled by lipid-dependent relocalization of the scaffold protein CKIP-1 and recruitment of the kinase CK2α to the plasma membrane, which seems to be essential for sufficient platelet biogenesis. Overall, this study provides a framework to understand how the MK lipidome is altered during maturation and the impact of MK membrane lipid remodeling on MK kinase signaling involved in thrombopoiesis.
PubMed: 38075556
DOI: 10.1038/s44161-023-00325-8 -
Cells Dec 2023The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood...
The bone marrow (BM) hematopoietic system (HS) gives rise to blood cells originating from hematopoietic stem cells (HSCs), including megakaryocytes (MKs) and red blood cells (erythrocytes; RBCs). Many steps of the cell-fate decision remain to be elucidated, being important for cancer treatment. To explore the role of Wnt/β-catenin for MK and RBC differentiation, we activated β-catenin signaling in platelet-derived growth factor b (Pdgfb)-expressing cells of the HS using a Cre-lox approach (Ctnnb1). FACS analysis revealed that Pdgfb is mainly expressed by megakaryocytic progenitors (MKPs), MKs and platelets. Recombination resulted in a lethal phenotype in mutants (Ctnnb1, Ctnnb1) 3 weeks after tamoxifen injection, showing an increase in MKs in the BM and spleen, but no pronounced anemia despite reduced erythrocyte counts. BM transplantation (BMT) of Ctnnb1 BM into lethally irradiated wildtype recipients (BMT-Ctnnb1) confirmed the megakaryocytic, but not the lethal phenotype. CFU-MK assays in vitro with BM cells of Ctnnb1 mice supported MK skewing at the expense of erythroid colonies. Molecularly, the runt-related transcription factor 1 (RUNX1) mRNA, known to suppress erythropoiesis, was upregulated in Ctnnb1 BM cells. In conclusion, β-catenin activation plays a key role in cell-fate decision favoring MK development at the expense of erythroid production.
Topics: Animals; Mice; beta Catenin; Megakaryocyte-Erythroid Progenitor Cells; Megakaryocytes; Proto-Oncogene Proteins c-sis; Thrombopoiesis
PubMed: 38067194
DOI: 10.3390/cells12232765 -
Life Science Alliance Feb 2024Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor...
Platelets display unexpected roles in immune and coagulation responses. Emerging evidence suggests that STING is implicated in hypercoagulation. STING is an adaptor protein downstream of the DNA sensor cyclic GMP-AMP synthase (cGAS) that is activated by cytosolic microbial and self-DNA during infections, and in the context of loss of cellular integrity, to instigate the production of type-I IFN and pro-inflammatory cytokines. To date, whether the cGAS-STING pathway is present in platelets and contributes to platelet functions is not defined. Using a combination of pharmacological and genetic approaches, we demonstrate here that megakaryocytes and platelets possess a functional cGAS-STING pathway. Our results suggest that in megakaryocytes, STING stimulation activates a type-I IFN response, and during thrombopoiesis, cGAS and STING are transferred to proplatelets. Finally, we show that both murine and human platelets contain cGAS and STING proteins, and the cGAS-STING pathway contributes to potentiation of platelet activation and aggregation. Taken together, these observations establish for the first time a novel role of the cGAS-STING DNA sensing axis in the megakaryocyte and platelet lineage.
Topics: Animals; Humans; Mice; Megakaryocytes; Signal Transduction; DNA; Cytokines; Nucleotidyltransferases; Interferon Type I
PubMed: 37993259
DOI: 10.26508/lsa.202302211 -
Nature Communications Nov 2023Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of...
Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.
Topics: Humans; Megakaryocytes; Thrombopoiesis; Erythropoiesis; Cell-Free Nucleic Acids; Blood Platelets; Erythroblasts; DNA
PubMed: 37985773
DOI: 10.1038/s41467-023-43310-2 -
JCI Insight Oct 2023Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both...
Patients with Down syndrome (DS), or trisomy 21 (T21), are at increased risk of transient abnormal myelopoiesis (TAM) and acute megakaryoblastic leukemia (ML-DS). Both TAM and ML-DS require prenatal somatic mutations in GATA1, resulting in the truncated isoform GATA1s. The mechanism by which individual chromosome 21 (HSA21) genes synergize with GATA1s for leukemic transformation is challenging to study, in part due to limited human cell models with wild-type GATA1 (wtGATA1) or GATA1s. HSA21-encoded DYRK1A is overexpressed in ML-DS and may be a therapeutic target. To determine how DYRK1A influences hematopoiesis in concert with GATA1s, we used gene editing to disrupt all 3 alleles of DYRK1A in isogenic T21 induced pluripotent stem cells (iPSCs) with and without the GATA1s mutation. Unexpectedly, hematopoietic differentiation revealed that DYRK1A loss combined with GATA1s leads to increased megakaryocyte proliferation and decreased maturation. This proliferative phenotype was associated with upregulation of D-type cyclins and hyperphosphorylation of Rb to allow E2F release and derepression of its downstream targets. Notably, DYRK1A loss had no effect in T21 iPSCs or megakaryocytes with wtGATA1. These surprising results suggest that DYRK1A and GATA1 may synergistically restrain megakaryocyte proliferation in T21 and that DYRK1A inhibition may not be a therapeutic option for GATA1s-associated leukemias.
Topics: Humans; Down Syndrome; GATA1 Transcription Factor; Leukemia, Megakaryoblastic, Acute; Thrombopoiesis
PubMed: 37906251
DOI: 10.1172/jci.insight.172851 -
Platelets Dec 2023Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal... (Review)
Review
Inherited thrombocytopenia (IT) is a group of hereditary disorders characterized by a reduced platelet count as the main clinical manifestation, and often with abnormal platelet function, which can subsequently lead to impaired hemostasis. In the past decades, humanized mouse models (HMMs), that are mice engrafted with human cells or genes, have been widely used in different research areas including immunology, oncology, and virology. With advances of the development of immunodeficient mice, the engraftment, and reconstitution of functional human platelets in HMM permit studies of occurrence and development of platelet disorders including IT and treatment strategies. This article mainly reviews the development of humanized mice models, the construction methods, research status, and problems of using humanized mice for the study of human thrombopoiesis.
Topics: Animals; Mice; Humans; Disease Models, Animal; Blood Platelets; Thrombopoiesis; Thrombocytopenia; Blood Platelet Disorders; Hematopoietic Stem Cell Transplantation
PubMed: 37849076
DOI: 10.1080/09537104.2023.2267676 -
Thrombosis Journal Oct 2023Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease characterized by increased platelet destruction and impaired thrombopoiesis. The changes in platelet...
BACKGROUND
Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease characterized by increased platelet destruction and impaired thrombopoiesis. The changes in platelet indices depend on the morphology and volume of platelets. Serum lipids have been found to affect platelet formation and activity in certain diseases, thus inducing the corresponding variation of platelet indices.
METHODS
Mendelian randomization (MR) analysis was performed based on databases. The clinical data from 457 ITP patients were retrospectively collected and analyzed, including platelet indices, serum lipids, hemorrhages and therapeutic responses.
RESULTS
MR analysis showed low high-density-lipoprotein-cholesterol (HDL-C), low apolipoprotein A-1, high triglyceride (TG) and high apolipoprotein B (ApoB) caused high platelet distribution width (PDW); high low-density-lipoprotein-cholesterol (LDL-C) increased mean platelet volume (MPV). In ITP, there were positive correlations between platelet count with TG, PDW with HDL-C and ApoB, and plateletcrit with TG and non-esterified fatty acid, and the correlation had gender differences. Bleeding scores were negatively correlated with cholesterol and LDL-C. LDL-C and homocysteine were risk factors for therapeutic responses.
CONCLUSIONS
Serum lipids, especially cholesterol were tightly correlated with platelet indices, hemorrhage and therapeutic effects in ITP patients. These results provide clinical references for the management of serum lipids, and highlight the necessity to further explore the relationship between lipids and pathogenesis of ITP.
TRIAL REGISTRATION
No: NCT05095896, October 14, 2021, retrospectively registered.
PubMed: 37833799
DOI: 10.1186/s12959-023-00551-x -
Cells Oct 2023Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we...
Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK's origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter (). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.
Topics: Mice; Animals; Thrombopoiesis; Microscopy; Blood Platelets; Megakaryocytes; Platelet Count
PubMed: 37830625
DOI: 10.3390/cells12192411 -
Scientific Reports Sep 2023Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development,...
Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, a comprehensive study on the microRNAs and their targets has not been undertaken. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, we identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. We knocked down each of these 15 microRNAs by the piggyback method and found knockdown of three microRNAs, mir-7148, let-7b, and mir-223 in adult zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. We also found enhanced thrombosis using arterial laser thrombosis assay in a group of zebrafish larvae after mir-7148, let-7b, and mir-223 knockdowns. These results suggested mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. We then explored miRWalk database for let-7b downstream targets and then selected those that are expressed in thrombocytes, and from this list based on their role in differentiation selected 14 genes, rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b that encode transcriptional regulators. The qRT-PCR analysis of expression levels of the above genes following let-7b knockdown showed changes in the expression of 13 targets. We then studied the effect of the 13 targets on thrombocyte production and identified 5 genes, irf5, tgif1, irf8, cebpa, and rorca that showed thrombocytosis and one gene, ikzf1 that showed thrombocytopenia. Furthermore, we tested whether mir-223 regulates any of the above 13 transcription factors after mir-223 knockdown using qRT-PCR. Six of the 13 genes showed similar gene expression as observed with let-7b knockdown and 7 genes showed opposing results. Thus, our results suggested a possible regulatory network in common with both let-7b and mir-223. We also identified that tgif1, cebpa, ikzf1, irf5, irf8, and ikzf1 play a role in thrombopoiesis. Since the ikzf1 gene showed a differential expression profile in let-7b and mir-223 knockdowns but resulted in thrombocytopenia in ikzf1 knockdown in both adults and larvae we also studied an ikzf1 mutant and showed the mutant had thrombocytopenia. Taken together, these studies showed that thrombopoiesis is controlled by a network of transcription regulators that are regulated by multiple microRNAs in both positive and negative manner resulting in overall inhibition of thrombopoiesis.
Topics: Adult; Humans; Animals; Thrombopoiesis; Zebrafish; Interferon Regulatory Factors; Thrombocytopenia; Thrombosis; MicroRNAs
PubMed: 37752184
DOI: 10.1038/s41598-023-42868-7