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Immunity Mar 2024Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical...
Emerging evidence has revealed a direct differentiation route from hematopoietic stem cells to megakaryocytes (direct route), in addition to the classical differentiation route through a series of restricted hematopoietic progenitors (stepwise route). This raises the question of the importance of two alternative routes for megakaryopoiesis. Here, we developed fate-mapping systems to distinguish the two routes, comparing their quantitative and functional outputs. We found that megakaryocytes were produced through the two routes with comparable kinetics and quantity under homeostasis. Single-cell RNA sequencing of the fate-mapped megakaryocytes revealed that the direct and stepwise routes contributed to the niche-supporting and immune megakaryocytes, respectively, but contributed to the platelet-producing megakaryocytes together. Megakaryocytes derived from the two routes displayed different activities and were differentially regulated by chemotherapy and inflammation. Our work links differentiation route to the heterogeneity of megakaryocytes. Alternative differentiation routes result in variable combinations of functionally distinct megakaryocyte subpopulations poised for different physiological demands.
Topics: Cell Differentiation; Thrombopoiesis; Megakaryocytes; Hematopoietic Stem Cells; Blood Platelets
PubMed: 38447571
DOI: 10.1016/j.immuni.2024.02.006 -
Research and Practice in Thrombosis and... Jan 2024A State of the Art lecture titled "Immune Attack on Megakaryocytes in ITP: The Role of Megakaryocyte Impairment" was presented at the International Society on Thrombosis...
A State of the Art lecture titled "Immune Attack on Megakaryocytes in ITP: The Role of Megakaryocyte Impairment" was presented at the International Society on Thrombosis and Haemostasis Congress in 2023. Immune thrombocytopenia (ITP) is an acquired autoimmune disorder caused by autoantibodies against platelet surface glycoproteins that provoke increased clearance of circulating platelets, leading to reduced platelet number. However, there is also evidence of a direct effect of antiplatelet autoantibodies on bone marrow megakaryocytes. Indeed, immunologic cells responsible for autoantibody production reside in the bone marrow; megakaryocytes progressively express during their maturation the same glycoproteins against which ITP autoantibodies are directed, and platelet autoantibodies have been detected in the bone marrow of patients with ITP. studies using ITP sera or monoclonal antibodies against platelet and megakaryocyte surface glycoproteins have shown an impairment of many steps of megakaryopoiesis and thrombopoiesis, such as megakaryocyte differentiation and maturation, migration from the osteoblastic to the vascular niche, adhesion to extracellular matrix proteins, and proplatelet formation, resulting in impaired and ectopic platelet production in the bone marrow and diminished platelet release in the bloodstream. Moreover, cytotoxic T cells may target bone marrow megakaryocytes, resulting in megakaryocyte destruction. Altogether, these findings suggest that antiplatelet autoantibodies and cellular immunity against bone marrow megakaryocytes may significantly contribute to thrombocytopenia in some patients with ITP. Finally, we summarize relevant new data on this topic presented during the 2023 International Society on Thrombosis and Haemostasis Congress. The complete unraveling of the mechanisms of immune attack-induced impairment of megakaryopoiesis and thrombopoiesis may open the way to new therapeutic approaches.
PubMed: 38525349
DOI: 10.1016/j.rpth.2024.102345 -
Cells Apr 2024Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing.... (Review)
Review
Platelets are the terminal progeny of megakaryocytes, primarily produced in the bone marrow, and play critical roles in blood homeostasis, clotting, and wound healing. Traditionally, megakaryocytes and platelets are thought to arise from multipotent hematopoietic stem cells (HSCs) via multiple discrete progenitor populations with successive, lineage-restricting differentiation steps. However, this view has recently been challenged by studies suggesting that (1) some HSC clones are biased and/or restricted to the platelet lineage, (2) not all platelet generation follows the "canonical" megakaryocytic differentiation path of hematopoiesis, and (3) platelet output is the default program of steady-state hematopoiesis. Here, we specifically investigate the evidence that in vivo lineage tracing studies provide for the route(s) of platelet generation and investigate the involvement of various intermediate progenitor cell populations. We further identify the challenges that need to be overcome that are required to determine the presence, role, and kinetics of these possible alternate pathways.
Topics: Animals; Mice; Blood Platelets; Cell Differentiation; Cell Lineage; Hematopoiesis; Hematopoietic Stem Cells; Megakaryocytes; Humans
PubMed: 38667319
DOI: 10.3390/cells13080704 -
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 -
Blood Cells, Molecules & Diseases Jan 2024Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without...
Thrombopoiesis is the production of platelets from megakaryocytes in the bone marrow of mammals. In fish, thrombopoiesis involves the formation of thrombocytes without megakaryocyte-like precursors but derived from erythrocyte thrombocyte bi-functional precursor cells. One unique feature of thrombocyte differentiation involves the maturation of young thrombocytes in circulation. In this study, we investigated the role of hox genes in zebrafish thrombopoiesis to model platelet production. We selected hoxa10b, hoxb2a, hoxc5a, hoxd3a, and hoxc11b from thrombocyte RNA expression data, and checked whether they are expressed in young or mature thrombocytes. We found hoxa10b, hoxb2a, hoxc5a, and hoxd3a were expressed in both young and mature thrombocytes and hoxc11b was expressed in only young thrombocytes. We then performed knockdowns of these 5 hox genes and found hoxc11b knockdown resulted in thrombocytosis and the rest showed thrombocytopenia. To identify hox genes that could have been missed by the above datasets, we performed knockdowns 47 hox genes in the zebrafish genome and found hoxa9a, and hoxb1a knockdowns resulted in thrombocytopenia and they were expressed in both young and mature thrombocytes. In conclusion, our comprehensive knockdown study identified Hoxa10b, Hoxb2a, Hoxc5a, Hoxd3a, Hoxa9a, and Hoxb1a, as positive regulators and Hoxc11b, as a negative regulator for thrombocyte development.
Topics: Animals; Thrombopoiesis; Zebrafish; Genes, Homeobox; Blood Platelets; Megakaryocytes; Thrombocytopenia; Mammals
PubMed: 37717409
DOI: 10.1016/j.bcmd.2023.102796 -
Biomolecules Apr 2024Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was... (Review)
Review
Thrombopoietin, the primary regulator of blood platelet production, was postulated to exist in 1958, but was only proven to exist when the cDNA for the hormone was cloned in 1994. Since its initial cloning and characterization, the hormone has revealed many surprises. For example, instead of acting as the postulated differentiation factor for platelet precursors, megakaryocytes, it is the most potent stimulator of megakaryocyte progenitor expansion known. Moreover, it also stimulates the survival, and in combination with stem cell factor leads to the expansion of hematopoietic stem cells. All of these growth-promoting activities have resulted in its clinical use in patients with thrombocytopenia and aplastic anemia, although the clinical development of the native molecule illustrated that "it's not wise to mess with mother nature", as a highly engineered version of the native hormone led to autoantibody formation and severe thrombocytopenia. Finally, another unexpected finding was the role of the thrombopoietin receptor in stem cell biology, including the development of myeloproliferative neoplasms, an important disorder of hematopoietic stem cells. Overall, the past 30 years of clinical and basic research has yielded many important insights, which are reviewed in this paper.
Topics: Thrombopoietin; Humans; Blood Platelets; Animals; Receptors, Thrombopoietin; Thrombopoiesis; Thrombocytopenia; Megakaryocytes
PubMed: 38672505
DOI: 10.3390/biom14040489 -
Nature Communications Mar 2024We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are...
We recently achieved the first-in-human transfusion of induced pluripotent stem cell-derived platelets (iPSC-PLTs) as an alternative to standard transfusions, which are dependent on donors and therefore variable in supply. However, heterogeneity characterized by thrombopoiesis-biased or immune-biased megakaryocytes (MKs) continues to pose a bottleneck against the standardization of iPSC-PLT manufacturing. To address this problem, here we employ microRNA (miRNA) switch biotechnology to distinguish subpopulations of imMKCLs, the MK cell lines producing iPSC-PLTs. Upon miRNA switch-based screening, we find imMKCLs with lower let-7 activity exhibit an immune-skewed transcriptional signature. Notably, the low activity of let-7a-5p results in the upregulation of RAS like proto-oncogene B (RALB) expression, which is crucial for the lineage determination of immune-biased imMKCL subpopulations and leads to the activation of interferon-dependent signaling. The dysregulation of immune properties/subpopulations, along with the secretion of inflammatory cytokines, contributes to a decline in the quality of the whole imMKCL population.
Topics: Humans; Megakaryocytes; Induced Pluripotent Stem Cells; Blood Platelets; Thrombopoiesis; MicroRNAs
PubMed: 38519457
DOI: 10.1038/s41467-024-46605-0 -
Recent Patents on Biotechnology 2024Immune thrombocytopenic purpura (ITP) is an autoimmune disorder determined by immune-mediated platelet demolition and reduction of platelet production. Romiplostim is a... (Review)
Review
Immune thrombocytopenic purpura (ITP) is an autoimmune disorder determined by immune-mediated platelet demolition and reduction of platelet production. Romiplostim is a new thrombopoiesis motivating peptibody that binds and stimulates the human thrombopoietin receptor the patent of which was registered in 2008. It is used to treat thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Romiplostim is a 60 kDa peptibody designed to inhibit cross-reacting immune responses. It consists of four high-affinity TPO-receptor binding domains for the Mpl receptor and one human IgG1 Fc domain. is a good host for the fabrication of recombinant proteins such as romiplostim. The expression of a gene intended in is dependent on many factors such as a protein's inherent ability to fold, mRNA's secondary structure, its solubility, its toxicity preferential codon use, and its need for post-translational modification (PTM). This review focuses on the structure, function, mechanism of action, and expressive approach to romiplostim in .
Topics: Humans; Purpura, Thrombocytopenic, Idiopathic; Escherichia coli; Patents as Topic; Blood Platelets; Thrombopoietin; Receptors, Fc; Recombinant Fusion Proteins
PubMed: 38282441
DOI: 10.2174/1872208317666230503094451 -
Journal of Thrombosis and Haemostasis :... Jun 2024Megakaryocytes (MKs) are polyploid cells responsible for producing ∼10 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the...
BACKGROUND
Megakaryocytes (MKs) are polyploid cells responsible for producing ∼10 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown.
OBJECTIVES
Here, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems.
METHODS
We manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets.
RESULTS
The genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34 cells.
CONCLUSION
These findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.
Topics: Megakaryocytes; Animals; Blood Platelets; Humans; Polyploidy; Thrombopoiesis; Mice, Knockout; Tubulin; Methylation; Histone-Lysine N-Methyltransferase; Mice, Inbred C57BL; Mice; Platelet Count
PubMed: 38537781
DOI: 10.1016/j.jtha.2024.03.010 -
Biological Research For Nursing May 2024Hematopoietic Stem Cell Transplant (HCT) is a potentially curative treatment for hematologic malignancies, including multiple myeloma. Biomarker investigation can guide...
Hematopoietic Stem Cell Transplant (HCT) is a potentially curative treatment for hematologic malignancies, including multiple myeloma. Biomarker investigation can guide identification of HCT recipients at-risk for poor outcomes. MicroRNAs (miRNAs) are a class of non-coding RNAs involved in the modulation and regulation of pathological processes and are emerging as prognostic and predictive biomarkers for multiple health conditions. This pilot study aimed to examine miRNA profiles associated with HCT-related risk factors and outcomes in patients undergoing autologous HCT. Patients eligible for autologous HCT were recruited and blood samples and HCT-related variables were collected. Differential expression analysis of miRNA was conducted on 24 patient samples to compare changes in miRNA profile in HCT eligible patients before and after transplant. Unsupervised clustering of differentially expressed ( .05) miRNAs pre- and post- HCT identified clusters of up- and down-regulated miRNAs. Four miRNAs (miR-125a-5p, miR-99b-5p, miR-382-5p, miR-145-5p) involved in hematopoiesis (differentiation of progenitor cells, granulocyte function, thrombopoiesis, and tumor suppression) were significantly downregulated post-HCT. Correlation analyses identified select miRNAs associated with risk factors (such as frailty, fatigue, cognitive decline) and quality of life pre- and post-HCT. Select miRNAs were correlated with platelet engraftment. Future studies should examine miRNA signatures in larger cohorts in association with HCT outcomes; and expand investigations in patients receiving allogeneic transplants. This will lead to identification of biomarkers for risk stratification of HCT recipients.
PubMed: 38819871
DOI: 10.1177/10998004241257847