An age-progressive platelet differentiation path from hematopoietic stem cells causes exacerbated thrombosis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Differentiation route determines the functional outputs of adult megakaryopoiesis.

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.

Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

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.

Chromatin modifier Hmga2 promotes adult hematopoietic stem cell function and blood regeneration in stress conditions.

The molecular mechanisms governing the response of hematopoietic stem cells (HSCs) to stress insults remain poorly defined. Here, we investigated effects of conditional knock-out or overexpression of Hmga2 (High mobility group AT-hook 2), a transcriptional activator of stem cell genes in fetal HSCs. While Hmga2 overexpression did not affect adult hematopoiesis under homeostasis, it accelerated HSC expansion in response to injection with 5-fluorouracil (5-FU) or in vitro treatment with TNF-α. In contrast, HSC and megakaryocyte progenitor cell numbers were decreased in Hmga2 KO animals. Transcription of inflammatory genes was repressed in Hmga2-overexpressing mice injected with 5-FU, and Hmga2 bound to distinct regions and chromatin accessibility was decreased in HSCs upon stress. Mechanistically, we found that casein kinase 2 (CK2) phosphorylates the Hmga2 acidic domain, promoting its access and binding to chromatin, transcription of anti-inflammatory target genes, and the expansion of HSCs under stress conditions. Notably, the identified stress-regulated Hmga2 gene signature is activated in hematopoietic stem progenitor cells of human myelodysplastic syndrome patients. In sum, these results reveal a TNF-α/CK2/phospho-Hmga2 axis controlling adult stress hematopoiesis.

The tubulin code in platelet biogenesis.

Blood platelets are small non-nucleated cellular fragments that prevent and stop hemorrhages. They are produced in the bone marrow by megakaryocytes through megakaryopoiesis. This intricate process involves profound microtubule rearrangements culminating in the formation of a unique circular sub-membranous microtubule array, the marginal band, which supports the typical disc-shaped morphology of platelets. Mechanistically, these processes are thought to be controlled by a specific tubulin code. In this review, we summarize the current knowledge on the key isotypes, notably ß1-, α4A- and α8-tubulin, and putative post-translational modifications, involved in platelet and marginal band formation. Additionally, we provide a provisional list of microtubule-associated proteins (MAPs) involved in these processes and a survey of tubulin variants identified in patients presenting defective platelet production. A comprehensive characterization of the platelet tubulin code and the identification of essential MAPs may be expected in the near future to shed new light on a very specialized microtubule assembly process with applications in platelet diseases and transfusion.

Inflammatory platelet production stimulated by tyrosyl-tRNA synthetase mimicking viral infection.

Platelets play a role not only in hemostasis and thrombosis, but also in inflammation and innate immunity. We previously reported that an activated form of tyrosyl-tRNA synthetase (YRSACT) has an extratranslational activity that enhances megakaryopoiesis and platelet production in mice. Here, we report that YRSACT mimics inflammatory stress inducing a unique megakaryocyte (MK) population with stem cell (Sca1) and myeloid (F4/80) markers through a mechanism dependent on Toll-like receptor (TLR) activation and type I interferon (IFN-I) signaling. This mimicry of inflammatory stress by YRSACT was studied in mice infected by lymphocytic choriomeningitis virus (LCMV). Using Sca1/EGFP transgenic mice, we demonstrated that IFN-I induced by YRSACT or LCMV infection suppressed normal hematopoiesis while activating an alternative pathway of thrombopoiesis. Platelets of inflammatory origin (Sca1/EGFP+) were a relevant proportion of those circulating during recovery from thrombocytopenia. Analysis of these "inflammatory" MKs and platelets suggested their origin in myeloid/MK-biased hematopoietic stem cells (HSCs) that bypassed the classical MK-erythroid progenitor (MEP) pathway to replenish platelets and promote recovery from thrombocytopenia. Notably, inflammatory platelets displayed enhanced agonist-induced activation and procoagulant activities. Moreover, myeloid/MK-biased progenitors and MKs were mobilized from the bone marrow, as evidenced by their presence in the lung microvasculature within fibrin-containing microthrombi. Our results define the function of YRSACT in platelet generation and contribute to elucidate platelet alterations in number and function during viral infection.

Daucosterol regulates JAK2-STAT3 signaling pathway to promote megakaryocyte differentiation.

Immune thrombocytopenia (ITP) is an autoimmune disease caused by the loss of immune tolerance to platelet autoantigens, resulting in reduced platelet production and increased platelet destruction. Impaired megakaryocyte differentiation and maturation is a key factor in the pathogenesis and treatment of ITP. Sarcandra glabra, a plant of the Chloranthaceae family, is commonly used in clinical practice to treat ITP, and daucosterol (Dau) is one of its active ingredients. However, whether Dau can treat ITP and the key mechanism of its effect are still unclear. In this study, we found that Dau could effectively promote the differentiation and maturation of megakaryocytes and the formation of polyploidy in the megakaryocyte differentiation disorder model constructed by co-culturing Dami and HS-5 cells. In vivo experiments showed that Dau could not only increase the number of polyploidized megakaryocytes in the ITP rat model, but also promote the recovery of platelet count. In addition, through network pharmacology analysis, we speculated that the JAK2-STAT3 signaling pathway might be involved in the process of Dau promoting megakaryocyte differentiation. Western blot results showed that Dau inhibited the expression of P-JAK2 and P-STAT3. In summary, these results provide a basis for further studying the pharmacological mechanism of Dau in treating ITP.

The role of PKC in X-ray-induced megakaryocyte apoptosis and thrombocytopenia.

Thrombocytopenia is a critical complication after radiation therapy and exposure. Dysfunction of megakaryocyte development and platelet production are key pathophysiological stages in ionizing radiation (IR)-induced thrombocytopenia. Protein kinase C (PKC) plays an important role in regulating megakaryocyte development and platelet production. However, it remains unclear how PKC regulates IR-induced megakaryocyte apoptosis. In this study, we found that pretreatment of PKC pan-inhibitor Go6983 delayed IR-induced megakaryocyte apoptosis, and inhibited IR-induced mitochondrial membrane potential and ROS production in CMK cells. Moreover, suppressing PKC activation inhibited cleaved caspase3 expression and reduced p38 phosphorylation levels, and IR-induced PKC activation might be regulated by p53. In vivo experiments confirmed that Go6983 promoted platelet count recovery after 21 days of 3 Gy total body irradiation. Furthermore, Go6983 reduced megakaryocyte apoptosis, increased the number of megakaryocyte and polyploid formation in bone marrow, and improved the survival rate of 6 Gy total body irradiation. In conclusion, our results provided a potential therapeutic target for IR-induced thrombocytopenia.

Megakaryocytic IGF1 coordinates activation and ferroptosis to safeguard hematopoietic stem cell regeneration after radiation injury.

BACKGROUND: Hematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression, which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury, while if and how the niche is reshaped and regulates HSC regeneration are poorly understood. METHODS: A mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number, distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry, immunofluorescence, colony assay and bone marrow transplantation, in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro, and was consolidated using megakaryocyte-specific knockout mice and transgenic mice. RESULTS: Megakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile, transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury, whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically, HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion, and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs, but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently, the delicate coordination between proliferation, mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly, punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury, representing a superior therapeutic approach for myelosuppression. CONCLUSIONS: Our study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.

Platelet and Megakaryocyte Roles in Innate and Adaptive Immunity.

Classically, platelets have been described as the cellular blood component that mediates hemostasis and thrombosis. This important platelet function has received significant research attention for >150 years. The immune cell functions of platelets are much less appreciated. Platelets interact with and activate cells of all branches of immunity in response to pathogen exposures and infection, as well as in response to sterile tissue injury. In this review, we focus on innate immune mechanisms of platelet activation, platelet interactions with innate immune cells, as well as the intersection of platelets and adaptive immunity. The immune potential of platelets is dependent in part on their megakaryocyte precursor providing them with the molecular composition to be first responders and immune sentinels in initiating and orchestrating coordinated pathogen immune responses. There is emerging evidence that extramedullary megakaryocytes may be immune differentiated compared with bone marrow megakaryocytes, but the physiological relevance of immunophenotypic differences are just beginning to be explored. These concepts are also discussed in this review. The immune functions of the megakaryocyte/platelet lineage have likely evolved to coordinate the need to repair a vascular breach with the simultaneous need to induce an immune response that may limit pathogen invasion once the blood is exposed to an external environment.

Diversity of Megakaryocytes.

Megakaryocytes are commonly known as large, polyploid, bone marrow resident cells that contribute to hemostasis through the production of platelets. Soon after their discovery in the 19th century, megakaryocytes were described in tissue locations other than the bone marrow, specifically in the lungs and the blood circulation. However, the localization of megakaryocytes in the lungs and the contribution of lung megakaryocytes to the general platelet pool has only recently been appreciated. Moreover, the conception of megakaryocytes as uniform cells with the sole purpose of platelet production has been challenged. Here, we review the literature on megakaryocyte cell identity and location with a special focus on recent observations of megakaryocyte subpopulations identified by transcriptomic analyses.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

Protein arginine methyltransferase 1 in the generation of immune megakaryocytes: A perspective review.

Megakaryocytes (Mks) in bone marrow are heterogeneous in terms of polyploidy. They not only produce platelets but also support the self-renewal of hematopoietic stem cells and regulate immune responses. Yet, how the diverse functions are generated from the heterogeneous Mks is not clear at the molecular level. Advances in single-cell RNA seq analysis from several studies have revealed that bone marrow Mks are heterogeneous and can be clustered into 3 to 4 subpopulations: a subgroup that is adjacent to the hematopoietic stem cells, a subgroup expressing genes for platelet biogenesis, and a subgroup expressing immune-responsive genes, the so-called immune Mks that exist in both humans and mice. Immune Mks are predominantly in the low-polyploid (≤8 N nuclei) fraction and also exist in the lung. Protein arginine methyltransferase 1 (PRMT1) expression is positively correlated with the expression of genes involved in immune response pathways and is highly expressed in immune Mks. In addition, we reported that PRMT1 promotes the generation of low-polyploid Mks. From this perspective, we highlighted the data suggesting that PRMT1 is essential for the generation of immune Mks via its substrates RUNX1, RBM15, and DUSP4 that we reported previously. Thus, we suggest that protein arginine methylation may play a critical role in the generation of proinflammatory platelet progeny from immune Mks, which may affect many immune, thrombotic, and inflammatory disorders.

Fusobacterium nucleatum Promotes Megakaryocyte Maturation in Patients with Gastric Cancer via Inducing the Production of Extracellular Vesicles Containing 14-3-3ε.

Fusobacterium nucleatum colonization contributes to the occurrence of portal vein thrombosis in patients with gastric cancer (GC). However, the underlying mechanism by which F. nucleatum promotes thrombosis remains unclear. In this study, we recruited a total of 91 patients with GC and examined the presence of F. nucleatum in tumor and adjacent non-tumor tissues by fluorescence in situ hybridization and quantitative PCR. Neutrophil extracellular traps (NETs) were detected by immunohistochemistry. Extracellular vesicles (EVs) were extracted from the peripheral blood and proteins in the EVs were identified by mass spectrometry (MS). HL-60 cells differentiated into neutrophils were used to package engineered EVs to imitate the EVs released from NETs. Hematopoietic progenitor cells (HPCs) and K562 cells were used for megakaryocyte (MK) in vitro differentiation and maturation to examine the function of EVs. We observed that F. nucleatum-positive patients had increased NET and platelet counts. EVs from F. nucleatum-positive patients could promote the differentiation and maturation of MKs and had upregulated 14-3-3 proteins, especially 14-3-3ε. 14-3-3ε upregulation promoted MK differentiation and maturation in vitro. HPCs and K562 cells could receive 14-3-3ε from the EVs, which interacted with GP1BA and 14-3-3ζ to trigger PI3K-Akt signaling. In conclusion, we identified for the first time that F. nucleatum infection promotes NET formation, which releases EVs containing 14-3-3ε. These EVs could deliver 14-3-3ε to HPCs and promote their differentiation into MKs via activation of PI3K-Akt signaling.

JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm.

Megakaryocytes (MKs) is an important component of the hematopoietic niche. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs. Using a murine model of MPN in which the human JAK2V617F gene is expressed in the MK lineage, we show that the JAK2V617F-bearing MKs promote hematopoietic stem cell (HSC) aging, manifesting as myeloid-skewed hematopoiesis with an expansion of CD41+ HSCs, a reduced engraftment and self-renewal capacity, and a reduced differentiation capacity. HSCs from 2-year-old mice with JAK2V617F-bearing MKs were more proliferative and less quiescent than HSCs from age-matched control mice. Examination of the marrow hematopoietic niche reveals that the JAK2V617F-bearing MKs not only have decreased direct interactions with hematopoietic stem/progenitor cells during aging but also suppress the vascular niche function during aging. Unbiased RNA expression profiling reveals that HSC aging has a profound effect on MK transcriptomic profiles, while targeted cytokine array shows that the JAK2V617F-bearing MKs can alter the hematopoietic niche through increased levels of pro-inflammatory and anti-angiogenic factors. Therefore, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging.

Megakaryocyte Secreted Factors Regulate Bone Marrow Niche Cells During Skeletal Homeostasis, Aging, and Disease.

The bone marrow microenvironment contains a diverse array of cell types under extensive regulatory control and provides for a novel and complex mechanism for bone regulation. Megakaryocytes (MKs) are one such cell type that potentially acts as a master regulator of the bone marrow microenvironment due to its effects on hematopoiesis, osteoblastogenesis, and osteoclastogenesis. While several of these processes are induced/inhibited through MK secreted factors, others are primarily regulated by direct cell-cell contact. Notably, the regulatory effects that MKs exert on these different cell populations has been found to change with aging and disease states. Overall, MKs are a critical component of the bone marrow that should be considered when examining regulation of the skeletal microenvironment. An increased understanding of the role of MKs in these physiological processes may provide insight into novel therapies that can be used to target specific pathways important in hematopoietic and skeletal disorders.

Heat shock protein 90 inhibitors induce cell differentiation via the ubiquitin-dependent aurora kinase A degradation in a MPLW515L mouse model of primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by immature megakaryocytic hyperplasia, splenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Our preclinical study had demonstrated that aurora kinase A (AURKA) inhibitor MLN8237 reduced the mutation burden of PMF by inducing differentiation of immature megakaryocytes. However, it only slightly alleviated splenomegaly, reduced tissue fibrosis, and normalized megakaryocytes in PMF patients of the preliminary clinical study. So enhancing therapeutic efficacy of PMF is needed. In this study, we found that AURKA directly interacted with heat shock protein 90 (HSP90) and HSP90 inhibitors promoted the ubiquitin-dependent AURKA degradation. We demonstrated that HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), normalized peripheral blood counts, improved splenomegaly, attenuated extramedullary hematopoiesis, decreased tissue fibrosis and reduced mutant burden in a MPLW515L mouse model of PMF. Importantly, both 17-AAG and 17-DMAG treatment at effective doses in vivo did not influence on hematopoiesis in healthy mice. Collectively, the study demonstrates that HSP90 inhibitors induce cell differentiation via the ubiquitin-dependent AURKA and also are safe and effective for the treatment of a MPLW515L mouse model of PMF, which may provide a new strategy for PMF therapy. Further, we demonstrate that combined therapy shows superior activity in acute megakaryocytic leukemia mouse model than single therapy.

Ormeloxifene, a nonsteroidal antifertility drug promotes megakaryocyte differentiation in leukemia cell line K562.

Ormeloxifene (ORM) (3,4-trans-2,2-dimethyl-3-phenyl-4-p-(ß-pyrrolidinoethoxy) phenyl-7-methoxychroman), world's first nonsteroidal selective estrogen receptor modulator approved for contraception in India has been shown to have potential anticancer activities. Here, we show that ORM can induce megakaryocyte and myeloid (granulocytic) but not erythroid differentiation in multipotent human myeloid leukemia cell line K562. We show that ORM at an IC50 of 7.5 µM can induce morphological changes similar to megakaryocytes in K562 cells. ORM led to increase in levels of megakaryocytic differentiation markers (CD41 and CD61) as well as key transcription factors GATA1 and AML1. We further show that ORM induces megakaryocytic differentiation in K562 cells through ERK activation and induction of autophagy in a fashion similar to other known inducers of megakaryocytic differentiation such as phorbol esters. In addition, as shown earlier, we yet again observed that ORM led to activation of caspases since their inhibition through pan-caspase inhibitor mitigated megakaryocytic differentiation as they led to significant decrease in CD41 and CD61. Because induction of megakaryocytic differentiation in K562 involves growth arrest and exit from cell cycle, we also observed an increase in levels of p21 and p27 with decrease in c-Myc protein levels in K562 cells treated with 7.5 µM ORM for 24 and 48 h, respectively. Taken together, these findings indicate that ORM can markedly induce megakaryocytic differentiation in K562 cells.

Clonal Megakaryocyte Dysplasia with Isolated Thrombocytosis Is a Distinct Myeloproliferative Neoplasm Phenotype.

INTRODUCTION: About 15% of people with a myeloproliferative neoplasm (MPN) are identified as MPN, unclassifiable using the 2016 WHO classification. METHODS: We tested whether persons with platelet concentration ≥450 × 10E+9/L, bone marrow megakaryocyte morphology typical of prefibrotic/early myelofibrosis (pre-MF), and no minor criteria of pre-MF should be classified as a distinct MPN subtype, clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). RESULTS: 139 subjects meet these criteria who we compared with primary myelofibrosis (PMF) including 402 with pre-MF and 521 with overt myelofibrosis. CMD-IT subjects were more likely female and younger. They had lower frequencies of JAK2V617F compared with persons with PMF (55% vs. 70%; p < 0.001) and higher frequencies of CALR mutations (37% vs. 17%; p < 0.001). They also had lower frequency of variations associated with JAK2V617F susceptibility, JAK2 46/1 (35% vs. 47%; p = 0.021), and VEGFA rs3025039 (12% vs. 17%; p = 0.030). Subjects with CMD-IT had lower incidences of thrombotic events compared with those with pre-MF (9.7% vs. 26%; p < 0.001) and longer survival (median, not reached vs. 23 years; HR = 0.34 (0.10, 0.30); p < 0.001). CONCLUSION: Our data indicate CMD-IT is a distinct MPN subtype and should be included in the classification of myeloid neoplasms.

Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration.

Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIbα, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.