Megakaryocytes Research Articles

Histone deacetylation-regulated cell surface Siglec-7 expression promoted megakaryocytic maturation and enhanced platelet-like particle release

BackgroundFunctioning as important hematologic cells for hemostasis, wound healing and immune defense platelets are produced before being released into the blood by cytoplasmic fragmentation at the end of the megakaryocyte (MK) differentiation, during which the involvement of both apoptosis and autophagy has been reported. Inhibitory sialic acid–binding immunoglobulin-like lectin-7 gene (Siglec-7) can be expressed on platelets and induce apoptosis on activation for uncharacterized function. ObjectiveWe aimed to investigate the regulatory mechanism for Siglec-7 activation along MK differentiation and its physiologic role during the MK maturation and platelet formation. MethodsBy using 2 well-established MK differentiation models (HEL and K562) and human primary CD34+ cell, we examined the upregulations of transcript and protein levels of Siglec-7 during MK differentiation, and the effect of Siglec-7 surface presence on MK differentiation and platelet-like particles (PLPs) release. ResultsWe show that both transcripts and surface Siglec-7 were elevated during MK differentiation, and the histone deacetylase 1 (HDAC1) acted as a negative regulator for Siglec-7 activation. By increasing Siglec-7 surface expression, we found that increased presence of Siglec-7 not only enhanced MK maturation but also the release of PLPs by activating caspase 3-dependent signaling, as evidenced in the observation of more CD41, polyploidy, and platelet factor 4 transcript formations. ConclusionIn this study, we demonstrated that Siglec-7 activation was subjected to epigenetic regulation, and the resulting induced expression of surface Siglec-7 played an important regulatory role in promoting MK differentiation, maturation, and PLP formation.

Targeting TLR2/Rac1/cdc42/JNK Pathway to Reveal That Ruxolitinib Promotes Thrombocytopoiesis.

Thrombocytopenia has long been considered an important complication of chemotherapy and radiotherapy, which severely limits the effectiveness of cancer treatment and the overall survival of patients. However, clinical treatment options are extremely limited so far. Ruxolitinib is a potential candidate. The impact of ruxolitinib on the differentiation and maturation of K562 and Meg-01 cells megakaryocytes (MKs) was examined by flow cytometry, Giemsa and Phalloidin staining. A mouse model of radiation-injured thrombocytopenia (RIT) was employed to evaluate the action of ruxolitinib on thrombocytopoiesis. Network pharmacology, molecular docking, drug affinity responsive target stability assay (DARTS), RNA sequencing, protein blotting and immunofluorescence analysis were applied to explore the targets and mechanisms of action of ruxolitinib. Ruxolitinib can stimulate MK differentiation and maturation in a dose-dependent manner and accelerates recovery of MKs and thrombocytopoiesis in RIT mice. Biological targeting analysis showed that ruxolitinib binds directly to Toll Like Receptor 2 (TLR2) to activate Rac1/cdc42/JNK, and this action was shown to be blocked by C29, a specific inhibitor of TLR2. Ruxolitinib was first identified to facilitate MK differentiation and thrombocytopoiesis, which may alleviate RIT. The potential mechanism of ruxolitinib was to promote MK differentiation via activating the Rac1/cdc42/JNK pathway through binding to TLR2.

Bifunctional effect of the inflammatory cytokine tumor necrosis factor α on megakaryopoiesis and platelet production

Background and ObjectivesPlatelets are affected by many factors, such as infectious or aseptic inflammation, and different inflammatory states may induce either thrombocytopenia or thrombocytosis. Tumor necrosis factor α (TNFα) is an important inflammatory cytokine that has been shown to affect the activity of hematopoietic stem cells. However, its role in megakaryocyte (MK) development and platelet production remains largely unknown. This study aimed to investigate the effects of TNFα on MK and platelet generation. Methods and ResultsThe ex vivo study with human CD34+ cells demonstrated that TNFα differentially modulated commitment toward the MK lineage. Specifically, a low concentration of 0.5 ng/ml TNFα promoted MK maturation, proplatelet formation, and platelet production, whereas a high concentration of 10 ng/ml or more TNFα exhibited a substantial inhibitory effect on MK and platelet production. The distinct effect of TNFα on MKs was mainly dependent on TNFα receptor 1. TNFα differentially regulated the MAPK–ERK1/2 signaling pathway and the cytoskeletal proteins cofilin and MLC2. The in vivo study with Balb/c mice indicated that low‐dose or high‐dose TNFα administration differentially affected short‐term platelet recovery after bone marrow transplantation. ConclusionsOur study revealed distinct roles for TNFα in megakaryopoiesis and thrombopoiesis and may provide new insights regarding the treatment for platelet disorders.

Occurrence and role of lung megakaryocytes in infection and inflammation.

Megakaryocytes (MKs) are large cells giving rise to platelets. It is well established that in adults, MKs develop from hematopoietic stem cells and reside in the bone marrow. MKs are also rare but normal constituents of the venous blood returning to the lungs, and MKs are found in the lung vasculature (MKcirc), suggesting that these cells are migrants from the bone marrow and get trapped in lung capillaries where the final steps of platelet production can occur. An unprecedented increase in the number of lung and circulating MKs was described in coronavirus disease 2019 (COVID-19) patients, suggesting that lung thrombopoiesis may be increased during lung infection and/or thromboinflammation. In addition to the population of platelet-producing intravascular MKs in the lung, a population of lung-resident megakaryocytes (MKL) has been identified and presents a specific immune signature compared to its bone marrow counterparts. Recent single-cell analysis and intravital imaging have helped us gain a better understanding of these populations in mouse and human. This review aims at summarizing the recent data on increased occurrence of lung MKs and discusses their origin, specificities, and potential role in homeostasis and inflammatory and infectious lung diseases. Here, we address remaining questions, controversies, and methodologic challenges for further studies of both MKcirc and MKL.

A New Role of NAP1L1 in Megakaryocytes and Human Platelets

Platelets (PLTs) are anucleate and considered incapable of nuclear functions. Contrastingly, nuclear proteins were detected in human PLTs. For most of these proteins, it is unclear if nuclear or alternatively assigned functions are performed, a question we wanted to address for nuclear assembly protein 1like 1 (NAP1L1). Using a wide array of molecular methods, including RNAseq, co-IP, overexpression and functional assays, we explored expression pattern and functionality of NAP1L1 in PLTs, and CD34+-derived megakaryocytes (MKs). NAP1L1 is expressed in PLTs and MKs. Co-IP experiments revealed that dihydrolipolylysine-residue acetyltransferase (DLAT encoded protein PDC-E2, ODP2) dynamically interacts with NAP1L1. PDC-E2 is part of the mitochondrial pyruvate-dehydrogenase (PDH) multi-enzyme complex, playing a crucial role in maintaining cellular respiration, and promoting ATP-synthesis via the respiratory chain. Since altered mitochondrial function is a hallmark of infectious syndromes, we analyzed PDH activity in PLTs from septic patients demonstrating increased activity, paralleling NAP1L1 expression levels. MKs PDH activity decreased following an LPS-challenge. Furthermore, overexpression of NAP1L1 significantly altered the ability of MKs to form proplatelet extensions, diminishing thrombopoiesis. These results indicate that NAP1L1 performs in other than nucleosome-assembly functions in PTLs and MKs, binding a key mitochondrial protein as a potential chaperone, and gatekeeper, influencing PDH activity and thrombopoiesis.

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.

The Immature Morphology of the Megakaryocytes Present in the Bone Marrow of Patients with Myelofibrosis Reflects Changes in the Frequency of Functionally Distinctive Subpopulations

We and others have demonstrated that the immature megakaryocytes (MK) observed in the bone marrow (BM) from patients with myelofibrosis (MF) play a causative role in the establishment of this disease [reviewed in 1]. By leveraging scRNA-seq and other methods, recent studies have identified the existence of at least three megakaryocyte subpopulations, each one exerting different functions [2-4]: the most prevalent is that of platelet producing MK, the second is niche MK (defined here as hematopoietic stem cell (HSC)-supporting for clarity) and the third is classified as immune MK. In addition, the human fetus contains a fourth subpopulation, also defined niche MK, characterized by high expression of extracellular matrix genes, such as COL1A1 and COL3A1, and by a TGF-β signature, which is responsible for shaping the extracellular matrix of the organs during development. Platelet producing MK fall into the high ploidy class, express genes related to thrombopoiesis and are characterized by the CD42b/ARNTL markers. HSC-supporting MKs are mostly >=8N with high expression of IGF1and PF4, which regulate the growth of HSCs, and are characterized by the CD42b/MYLK4 markers. Immune MK are low ploidy with signatures of inflammatory response and myeloid leukocyte activation genes and are characterized as CD41/LSP1 cells. Lastly, niche MK, which are absent in adult BM, are characterized as CD42b/Col I and/or Col III cells. We hypothesize that the immature morphology of the MK from the BM of MF patients flags changes in the relative frequencies of the different MK subpopulations and induction of the formation of niche MK, originally identified in the fetus. To test this hypothesis, we performed extensive confocal microscopy studies with MK-specific markers on the BM from three patients with MF (and three patients with essential thrombocythemia, ET, or with other malignancies but no fibrosis, non-MF, as control) and of the BM from Gata1low mice before (young) and after (old) onset of MF, using age matched wild type (WT) littermates as control (three mice/group). First we confirmed that the BM from MF patients contains greater numbers of MK than that from ET and non-MF patients and that these MF MK have an immature morphology and contain lower levels of GATA1 compared to controls (Fig. 1). We then demonstrated that MF MK are engaged in greater numbers in neutrophils emperipolesis associated with Netosis (Fig. 1), two events predicted to be triggered by the high bioavailability of interleukin-8 in the BM microenvironment of MF patients. Third, we observed that platelet MK (CD42b+ARNTL+) are present in the BM from MF, ET and non-MF patients and from that of WT and Gata1low mice of all ages. By contrast, HSC-supporting MK (CD42b+MYLK4+) are observed in the BM from ET but not in that from MF. They are also observed in the BM from WT mice (both young and old) and from young Gata1low mice, but not in that from old Gata1low mice. Immune MK (CD42b+LSP1+) are instead observed in the BM from ET and non-MF patients but not in MF patients and in the BM from young and old WT mice and in that from young Gata1low mice but not in that from old Gata1low mice. Lastly, great numbers of the CD42b MK in the BM from MF patients, but not in that from ET and non-MF, contain Col I and III (Fig. 1). These cells are also present in the BM from old Gata1low mice but not in their age-matched WT littermates (data not shown) and may represent niche MK the differentiation of which is reactivated by the high TGF-β bioavailability in the BM from MF. In conclusion, these results confirm the hypothesis that changes in frequency of MK subpopulations, rather than in MK maturity state per se, plays a causative role in the development of MF.

Actin Bundling Protein L-Plastin Regulates Megakaryocyte Membrane Rigidity and Platelet Spreading

An adequate number of normally functioning platelets is required for normal hemostasis, while thrombocytopenia and platelet dysfunction are common causes of pathologic bleeding. Platelets are released into the blood from proplatelets (PPs) that extend from megakaryocyte (MK) intracellular invaginated membrane system (IMS) that is continuous with the plasma membrane. Tubulin provides the motor for PP extension, and actin reorganization is believed to be critical for MK migration and PP branching. However, the role for actin in PP formation (PPF) is less well understood. The initiation of PPF requires podosomes - actin-based structures with an F-actin core surrounded by a ring of adhesion molecules (e.g., integrins) and many actin-binding proteins. We have been intrigued by how podosomes form and how the IMS is able to penetrate the dense cortical actin cytoskeleton to form PPs. We recently showed that human MKs and platelets express a novel actin bundling protein, L-plastin. L-plastin levels were inversely correlated with platelet number, and its expression was down-regulated during MK maturation. L-plastin knockdown in cultured human CD34+ derived MKs caused increased PP, IMS, and podosome formation. These data indicate L-plastin is a negative regulator of MK platelet production. To assess the in vivo effects of L-plastin, we have investigated platelet production and function in vivo using L-plastin knockout (KO) mice (Lcp1-/-). Compared to wildtype (WT) littermate controls, Lcp1 KO mice showed both significantly increased platelet counts and increased PPF from cultured murine bone marrow MKs. Elegant work in both HUVECs and epithelial cells has shown high membrane proximal F-actin (MPA) prohibits membrane protrusions, and lowering MPA density is required to sensitize areas for membrane protrusion (Bisaria, Science 2020). We hypothesize that normal MK levels of L-plastin inhibits PPF in less mature MKs by F-actin bundling, which raises the MPA. Conversely, lower L-plastin levels result in lower MPA and enabling PP protrusion. Enhanced L-plastin-mediated F-actin bundling is expected to increase membrane stiffness, so we measured MK membrane stiffness at single cell level using a state-of-the-art Biomembrane Force Probe (BFP). MKs from KO mice showed 68% reduction in membrane stiffness as compared to WT controls (p<0.0001) (Fig 1). Ultrastructure of MKs also revealed increased IMS in Lcp1-/- MKs. Preliminary data suggests that L-plastin deficiency also associated with increased MK podosome formation in vitro, which is consistent with our findings in human CD34+ derived MKs. Although L-plastin levels decrease during terminal MK differentiation, they persist at moderate levels in platelets. Platelet integrin activation, granule release and clot retraction also depend on actin reorganization, but L-plastin function in platelets has not been studied. We observed no difference between WT and Lcp1 KO platelets for measures of inside-out integrin signaling (activation-induced JON/A binding or P-selectin expression). However, when plated on fibrinogen and stimulated with thrombin, platelets from KO mice showed significantly increased spreading compared to WT mice (Fig 2). A similar trend was observed for platelet clot retraction. Taken together these results show that L-plastin regulates MK membrane stiffness, which likely inhibits PPF. L-plastin also appears to participate in platelet outside-in, but not inside-out, integrin signaling. On-going studies are determining whether integrin beta3 is the molecular link between these two findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SETD2 Regulates Nuclear Division to Achieve Polyploidization in Megakaryocyte

Megakaryocytes (MKs) undergo a fascinating endomitosis process to become naturally large multilobulated, polyploid nuclei cells that give rise to platelets. Two key steps involving in this process are the controlled failure in cytokinesis (cytoplasm division) and karyokinesis (nuclear division). The cytokinesis defect has been reported to be regulated by factors related to abnormal actomyosin ring and cleavage furrow formation such as RhoA, MYH9 and Rac1. In contrast, it remains largely unknown how nuclear division defect is regulated during MK endomitosis. Using a mouse model and an in vitro differentiation system, we found SETD2 expression markedly declined when MKs underwent polyploidization. Knockdown of SETD2 increased the high ploidy of primary bone marrow (BM) murine MKs, as well as MKs derived from cultured human cord-blood CD34+ cells and MEG-01 cells. Furthermore, the higher ploidy of MKs was accompanied by the increase of platelet formation both in the mouse model and the in vitro differentiation system. With forced overexpression of SETD2, the proportion of high ploidy MKs (≥8N) was remarkably decreased. These data suggest that SETD2, an α-tubulin methyltransferase, negatively regulates polyploidization during megakaryopoiesis. To investigate how SETD2 regulates endomitosis, we employed live-cell imaging to monitor the whole process of endomitosis at the single-cell level by observing the spindle geometry. We found SETD2 deficient MKs were capable of completing the endomitotic cycle, allowing cells to reach a higher ploidy in a significantly shorter time compared to control cells (2.9 hours vs. 4.9 hours). In comparison, overexpression of SETD2 significantly increased endomitosis cycle time compared to control conditions (5.2 hours vs. 4.3 hours). These results show that SETD2 deficiency accelerates the endomitotic cell cycle. Moreover, loss of SETD2 increased the percentage of cells within the presence of nucleoplasmic bridges (NPBs), a thin DNA link referred as a characteristic structure for karyokinesis defect. These results suggest that SETD2 may regulate endomitosis through facilitating the defect of karyokinesis. During karyokinesis, microtubules localized near spindle poles are need to be depolymerized as force-generating apparatus to separate nuclei. Thus, we measured the amount of polymerized and depolymerized microtubule forms in the presence and absence of SETD2. We found that loss of SETD2 decreased the proportion of the depolymerized form of tubulin in spindles, while SETD2 overexpression increased depolymerized microtubule forms. These data suggest that loss of SETD2 promotes the defect of karyokinesis by reducing depolymerized microtubule state. Mechanistically, as SETD2 could trimethylate α-tubulin at lysine 40 (α-tubK40me3), we found that the levels of α-TubK40me3 near spindle poles were inversely correlated with the degree of karyokinesis defect and MK polyploidization. Moreover, SETD2-deficiency resulted in an absence of α-TubK40me3 with an increase of the MK ploidy. And this ploidy increase was largely reversed by overexpressing a mutant tubulin protein that mimics the α-tubK40me3. Together, these data indicate that SETD2-mediated α-TubK40me3 regulates MK polyploidization by influencing microtubule dynamics. In summary, our study reveals that SETD2 is an important, novel regulator of MK endomitosis by regulating α-TubK40me3 to influence microtubule dynamics. These findings help fill current knowledge gaps in the mechanism of the karyokinesis defect during MK endomitosis. Targeting SETD2 may improve high ploidy MKs and platelet production that could promote the regeneration of platelets in vitro and help treat megakaryocytic dysplasia diseases with abnormal polyploidization in the future.

Fusion of uPA to Anti-aIIbb3 Scfv Facilitates Its Uptake By in Vitro-Generated CD34+ Megakaryocytes (MKs) to Promote Targeted Fibrinolysis

We wish to develop a clinically relevant strategy to deliver anti-thrombotic urokinase plasminogen activator (uPA)-carrying platelets (uPA-PLTs) as a targeted thromboprophylaxis that can be administered to patients with a concurrent high risk of bleeding. This intent was based on our previous data showing that transgenic mice, ectopically expressing uPA in megakaryocytes (MKs), released PLTs, containing uPA in α granules, that were effective as a thromboprophylaxis agent (PMID12689937). We faced two major challenges in further developing uPA-containing PLTs for clinical application: (1) How to get uPA into PLTs as human Plts do not take up uPA, and (2) α-granular uPA led to proteolysis of other α-granule proteins. To diminish degradation of α-granular proteins by uPA, we generated a plasmin-insensitive, but thrombin-activatable, truncated uPA mutant (uPA-T, bottom, Fig. 2). We found that both native single-chain uPA (scuPA) and uPA-T can be taken up by human MKs into the same α granules whereby they co-localize with endocytosed Factor V (FV) and/or fibrinogen (FBGN), as evidenced by confocal microscopy, suggesting that the uPA variants use the same endocytic receptor pathway, also utilized by FV and FBGN (Fig. 1). To delineate what receptor system(s) mediate uPA uptake by CD34+ MK, we found that FV (known to be partially internalized via low-density lipoprotein receptor-related protein 1 (LRP1) competed with both uPA variants for uptake, and that uptake of uPA variants and FV was partially inhibited by the LRP1 antagonist, receptor-associated protein (RAP), further supporting that LRP1 mediates internalization of the uPAs. We also found that uPA and FBGN uptake was partially inhibited by αIIbβ3 integrin antagonist ReoPro. Since we observed that LRP1 protein expression and extent of uPA uptake by MKs declines upon maturation, whereas αIIbβ3 expression does not, we tested if targeting of uPA to αIIbβ3 would facilitate its uptake by CD34+ MKs. We generated a chimeric prodrug composed of a single-chain version of the variable region (scFv) of an anti-αIIbβ3 monoclonal antibody fused to uPA-T (PLT/uPA-T, bottom Fig. 2) which binds human αIIbβ3 on both quiescent and activated PLTs, and is enzymatically activated specifically by thrombin. We have previously shown that in two murine injury models, PLT/uPA-T did not lyse preexisting clots, while it concurrently prevented the development of nascent thrombi (PMC4731161). However, direct infusion of PLT/uPA-T in mice caused significant thrombocytopenia. Now, we found that CD34+ MK internalized PLT/uPA-T into the same α granules of MKs, but with significantly higher efficiency than uPA-T protein (top Fig. 2) which will optimize loading of MKs by uPA-T for subsequent use as a source of uPA-PLTs. We found that scuPA-loaded platelets released from infused MKs prevent thrombus formation in a Rose Bengal photochemical injury model. These studies were done using immunocompromised NOD-scid IL2rγnull (NSG) mice homozygous for VWFR1326H (a VWF mutation switching binding specificity from mouse to human GPIb/IX). NSG/VWFR1326H mice had impaired clotting after vascular injury compared to NSG mice unless infused with human PLTs or MKs. We found that MKs that endocytosed uPA-T can also prevent thrombi formation in this model. Future studies will test whether PLTs released from PLT/uPA-T-loaded MKs would further enhance efficacy of this thromboprophylactic strategy, while avoiding thrombocytopenia, and can become a point-of-care thromboprophylaxis therapeutic in the clinical setting of high-risk of developing thrombi with concurrent concerns of inducing significant bleeding. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

UBASH3B knockout Downregulates STAT1 Mediated Interferon Signalling and Megakaryocyte Related Genes in a JAK2 V617F Positive MPN Model

Interferon (IFN) signalling via STAT1 plays an important role in the pathogenesis and treatment of myeloproliferative neoplasms (MPN). Single cell sequencing approaches have implicated megakaryocyte (MKC) biased stem cells with enhanced intrinsic activation of IFN signalling pathways in MPN patients as critical mediators of disease initiation and maintenance. UBASH3B is a histidine phosphatase and has been identified as a potential regulator of malignant haematopoiesis and as a mediator of JAK/STAT1 IFN signalling in lymphoid cells. RNA sequencing (RNA-seq) data in JAK2 V617F positive UKE-1, HEL (both own lab) and SET2 (GSE91062) cells demonstrate that UBASH3B is significantly downregulated following administration of the JAK inhibitor ruxolitinib (RUX). We observed that UBASH3B expression in CD34+ cells in patients with primary myelofibrosis (GSE53482) is significantly enhanced but reduced in patients with essential thrombocythaemia(ET) and polycythaemia (GSE103237) and hypothesized that UBASH3B may contribute to MPN pathogenesis. UKE-1 is a JAK2 V617F positive primitive haematopoietic cell line derived from an ET patient. It shows features of intrinsic IFN signalling and expresses genes related to MKC development and differentiation providing a useful model of MPN stem cell behaviour. CRISPR-Cas9 mediated KO of UBASH3B was performed in UKE-1 cells. A ribonucleoprotein complex of guide RNA and Cas9 protein was nucleofected into cells. Individual colonies were assessed for UBASH3B status using western blotting and Sanger sequencing. Four distinct, healthy KO clones were established for the UKE-1 cell line and compared to the original wildtype cell population. RNA-seq and phospho-proteomic analysis were undertaken in two KO clones. Extracted RNA was sequenced on Illumina platforms with a minimum of 45 million individual paired reads per sample. Differential gene expression was analysed using DESEQ2. Whilst, matched cell lysates underwent tandem mass tagging, high pH fractionation, and phospho-peptide enrichment was undertaken prior to liquid chromatography-mass spectrometry for total and phospho-proteomic analysis. RNA-seq and total proteomic analysis were consistent in demonstrating a significant downregulation of STAT1 (Log2FC-0.79, FDR 9.03E-49) and STAT2 (Log2FC -0.70, FDR 9.05E-20) with over-enrichment analysis identifying downregulation of type 1 IFN signalling pathways and pathways related to MKC and platelet function as shown in Figure 1. The significant downregulation of genes associated with platelet function, MKC development and differentiation included GATA1, GP1BA, ITGA2B, SELP, VWF and genes identified as mediators of fibrosis in MPN as demonstrated in Figure 2. Targeted real time PCR for STAT1, GP1BA and LTPB1 confirmed the same transcriptional patterns were present in both additional knockout clones. Knockout clones show a diminished response to exogenous IFN alpha administration consistent with downregulation of these pathways. Consistently reduced proliferation is observed in the KO clones by a variety of methodologies. Comparison to the transcriptional response in RUX treated UKE-1 cells demonstrates that at 24 hours post RUX treatment significant STAT1 downregulation is observed but not accompanied by the same pattern of down-regulation of MKC and fibrosis associated genes. The same was seen in cells persistently treated with high dose RUX for greater than 5 weeks. These results show a significant downregulation of IFN signalling in UBASH3B KO cells linked to a significant downregulation of MKC associated genes further highlighting the link observed in MPN stem cells. They implicate UBASH3B as a potential mediator of IFN signalling in a JAK2 V617F context and support a hypothesis that downregulating IFN signalling in MPN stem cells may have disease modifying potential by altering differentiation and pro-fibrotic potential. RUX treatment downregulates STAT1 without activating the same transcriptional programme associated with MKC gene downregulation and this may help explain the limited disease modifying effect of RUX observed in patients. This work has generated an important KO cell line model which will permit the molecular characterisation of differential STAT1/IFN pathway activation and allow further study to understand the regulation of MKC differentiation and pro-fibrotic gene transcription observed in MPN stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Approach to 3D Spatial Mapping of the Human Hematopoietic Microenvironment in Normal and Diseased Bone Marrow

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. Important cellular components of the human (HME) include the bone marrow (BM) stromal cells (Li, BioRxive 2022) and other cell types, such as vascular endothelial cells (ECs), and megakaryocytes (MKs). Perturbations of the HME can affect normal and diseased hematopoiesis. In Philadelphia-negative myeloproliferative neoplasms (MPNs), which include Essential Thrombocythemia (ET), Polycythemia Vera (PV), Primary Myelofibrosis (PMF), and its precursor, pre-fibrotic MF (pre-PMF), the HME is of critical importance for disease development and progression (Mead, Blood 2017). However, despite this important role, little is known about the spatial organization of the different HME elements in MPNs. Therefore, we aimed to develop a novel method of immunofluorescence (IF) multiplexing with markers for HME and hematopoietic cells based on sequential staining and bleaching in combination with scanning IF microscopy and standard imaging analysis (Fig.1). Herein we show that this approach allowed us to generate 3D reconstitutions of standard biobank-stored BM samples from MPN patients and age-matched hematopoietically normal controls. Furthermore, we demonstrate how the free and open-source 3D computer graphics software toolset Blender (Stichting Blender Foundation, Amsterdam) can be utilized to analyze cell distributions and spatial relationships in 3D. BM biopsies from patients with MPN and controls were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Twelve subsequent slides per sample were stacked to create three-dimensional BM reconstructions with the imaging program Arivis Vision 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in Blender. We recapitulated the BM architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed considerable differences between the different MPN types, especially concerning megakaryocyte (MK) morphology and distribution, as well as CD271 staining density and CD271+ cell numbers. Whereas ET and pre-PMF were comparable to their paired normal control, we found that PMF and especially PV samples showed considerably increased numbers and densities of CD271+ cells. In PMF the generated MSC objects formed a thick fibrillary structure reminiscent of classic fibrosis staining that was particularly dense close to vessels, bone surfaces, and MKs. Measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells (HSPC) to vessels and bone structures, respectively, revealed the most pronounced differences in PV, with median distances of PV-MKs to vessels being significantly lower than in controls (PV 7.62 µm vs 8.20 µm control, p<0.05). Also, the median distance in PV of HSPCs to vessels (2,88 µm) and bones (52.23 µm) was less than in the controls (46.31 µm and 236.7 µm, respectively; p<0.01). Taken together, we report a novel sequential staining and bleaching technique for multi-color analysis of normal and diseased BMs. The three-dimensional reconstruction technique developed herein revealed important aspects of the spatial HME composition in MPNs and normal BMs and provides a platform for a deeper investigation of BM pathophysiology, for example by increasing numbers of antibodies and including visualization of the expression of relevant genes and proteins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Tumor Necrosis Factor from Leukemic Microenvironment Provides Non-Leukemic Hematopoietic Stem Cells with Megakaryocyte Lineage Bias

Background: Acute myeloid leukemia (AML) is characterized by proliferation of malignant cells and impairment of normal hematopoiesis, leading to the severe bone marrow failure. Although genetic abnormalities and pathogenesis of AML are now investigated in detail, the precise mechanism impairing normal hematopoiesis in AML is not fully elucidated yet. Hematopoietic stem cells (HSC) retain the self-renewal capacity together with multipotent differentiation potential into all mature blood cells, playing a major role in maintaining the normal hematopoiesis. The ability of HSC is maintained by complex network of intrinsic factors and external stimulation from microenvironment called as HSC niche. According to these backgrounds, we hypothesized that the impairment of normal hematopoiesis in AML is caused by abnormal function of non-leukemic (nl) HSC. To verify this hypothesis, we isolated nl-HSC and HSC niche cells from MLL-AF9-induced AML mice and further analyzed their function and gene expression profiles. Results: The frequencies of nl-HSC/hematopoietic progenitor fraction (Lin-cKit+Sca1+ cells, HSPC) were significantly increased at the initiation phase of leukemic proliferation in AML mice (leukemic cells : 10-30% in BM)（normal: 0.76+/-0.34%, AML: 2.07 +/-0.93 %, p<0.001）. Surface expression level of CD41 was also upregulated (normal: 15.1+/- 0.526%, AML: 25.4+/- 9.28%, p<0.05). Interestingly, the results of in vitro culture of nl-HSPC demonstrated that they proliferated more efficiently than HSPC from normal mouse BM, and that they tended to differentiate into megakaryocyte (MgK) lineage. Gene set enrichment analysis revealed that MgK-related gene sets and several inflammatory cytokines signaling pathways, including tumor necrosis factor (TNF) pathway, were statistically enriched in nl-HSC. These data suggested that nl-HSC/HSPC retain the MgK-biased character. On the other hands, the gene expression profiles of perivascular stromal cells (ICAM1+CD31-CD45-TER119-cells) in AML mice demonstrated the significantly decreased expression of HSC-supporting factors (e.g., lpl, ptn, rspo2, angptl4, kitl, cxcl12), when compared with those in normal mice. These data suggested that the changes of multiple external humoral factors from leukemic environment induce functional alternation of nl-HSC. To further investigate the role of inflammatory humoral factors in HSC self-renew and differentiation, we next utilized polyvinyl alcohol (PVA)-based ex vivo HSC expansion system and focused on the role of TNF signaling. In the analysis of highly purified normal HSC (CD150+CD48-CD34-cKit+Sca1+Lin-cells) with PVA, TNF alpha increased both proliferation and MgK differentiation. UMAP analysis of single cell RNA sequence data for ex vivo-expanded HSCs with TNF alpha showed the increased frequencies of the clusters expressing MgK-related genes (e.g., itga2b and gp1ba) and relatively decreased immature HSC related genes (e.g., hlf and procr). The results of competitive repopulation assay using ex-vivo expanded normal HSC with TNF alpha stimulation in HSC promoted myeloid differentiation of HSC, rather than self-renew and expansion of HSC. Conclusion: Our data suggested that TNF alpha from leukemic microenvironment may play a key role for the driver signaling of HSCs to MgK, eventually resulting in the impairment of normal HSC maintenance.

Human Bone Marrow Organoids Enable the Study of Hematopoietic Cell-Stromal Interactions and Support the Survival of Malignant Cells from Patients

Background: A lack of scalable experimental platforms that adequately recapitulate human bone marrow (BM) has hampered the study of normal and malignant hematopoiesis, as well as the validation of novel therapies. Although organoid technologies are rapidly advancing research in other fields, organoids that models the complexity of native BM have not yet been reported. Our aim was to engineer human induced pluripotent stem cell (iPSC)-derived organoids with homology to human BM, and to assess their utility for modelling blood and BM disorders. Methods: A directed differentiation protocol giving rise to mesenchymal stromal cells (MSCs), endothelial cells (ECs) and myeloid cell subtypes was developed and characterized by single cell RNA-sequencing, multi-modal imaging and functional studies. Architectural and transcriptional homology to human BM was assessed, and the utility of the system as a model for normal and perturbed hematopoiesis was determined, including the ability to support engraftment and survival of primary cells from healthy donors and patients with blood cancers. Results: Hydrogel composition and growth factor supplementation were optimised to support the generation of self-organizing, vascularised 3D cultures with cellular, molecular and architectural homology to the central myelopoietic perivascular niche. Organoids were composed of MSCs, fibroblasts, sinusoidal ECs, as well as myelomonocytic, erythroid, megakaryocytic, and eo-baso-mast cells (Fig. 1A). 3D imaging revealed a dense network of lumen-forming vessels invested with MSCs/fibroblasts (Fig. 1A), with perivascular megakaryocytes (MKs) and myeloid cells migrating to the sinusoidal lumens. Comparison to primary human tissue revealed immunophenotypic and transcriptional specialization of ECs and MSCs to resemble BM sinusoidal endothelium and MSCs respectively. Endogenous production of stem cell factor, GM-CSF, FLT3L, CD40L and other hematopoiesis-promoting cytokines and chemokines by organoids was confirmed at the protein level. Strong autocrine and paracrine receptor-ligand interactions were detected between hematopoietic cells and their stroma, recapitulating regulatory interactions occurring in the BM niche, including CXCL12, NOTCH1, FGF, KITL and ANGPT2, PDGF and TGFβ and interleukin signalling pathways. Having demonstrated homology to human BM and confirmed the capacity of our system to generate supporting growth factors, we hypothesized that the organoids could provide a platform for the growth and maintenance of primary adult BM cells, and mimic cancer-induced niche remodelling. Seeding of organoids with CD34+ cells from healthy donors and patients with myelofibrosis resulted in the rapid engraftment, proliferation and differentiation of donor cells within the organoids, as well as the maintenance of a CD34+, quiescent stem/progenitor population. Myelofibrosis CD34+ cells, but not cells from healthy donors, induced fibrotic remodelling of the organoid niche (collagen deposition and myofibroblast activation). Fibrosis was inhibited by treatment with JQ1, a potent BET bromodomain inhibitor, but not ruxolitinib, a JAK1+2 inhibitor widely used in myelofibrosis treatment that, while reducing splenomegaly and symptoms in a majority of patients, does not typically improve fibrosis. Dose-dependent induction of hallmarks of fibrosis in organoids also occurred following TGFβ treatment without patient cells, enabling the robust screening of candidate inhibitors of fibrosis. We next assessed whether the organoids could support survival and proliferation of hematologic cancer cells that are particularly challenging to maintain ex vivo. We found significantly improved viability and proliferation of cells from patients with myeloid and lymphoid malignancies including multiple myeloma and acute lymphoblastic leukemia over 12 days of culture as compared to both liquid culture and the previous gold standard, 3D co-culture with human BM MSCs, and the cells maintained their immunophenotype. Conclusion: We describe a novel technology for the study of normal and perturbed hematology that we hope will be transformative in mechanistic studies of hematopoiesis and in modelling BM disorders (Fig. 1B), and accelerate target discovery, validation and translation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Role of Heparan Sulfate 6-O-Endosulfatases in Hematopoiesis

Heparan sulfate proteoglycans (HSPGs) are expressed at the cell surface and in the extracellular matrix, including within the bone marrow microenvironment. Heparan sulfate (HS) is the glycosaminoglycan side chain of HSPGs, and these side chains bind to a myriad of growth factors, cytokines, chemokines, and extracellular matrix molecules, mediating a diverse range of biological functions. It is well established that HS-protein interactions critically depend on the amount and position of 6-O-sulfate groups, which form binding sites for proteins. The HS 6-O-endosulfatases, the Sulfs (Sulf1 and Sulf2), remove 6-O-sulfates from specific HS intra-chain sites and are the only known enzymes that can modify HS sulfation status extracellularly. A gain or a loss of HS 6-O-sulfation can alter the outcome of HS-ligand interactions, such as with Wnt ligands, fibroblast growth factors (FGF), and transforming growth factor (TGF)-β1. Monocytes and macrophages express the Sulfs, predominantly Sulf2, and their secretion of the Sulfs can influence HS sulfation status in their surrounding microenvironments. The objective of this study was to examine the impact of myeloid lineage Sulf deletion on hematopoiesis. For this investigation, we generated a murine model of Sulf deletion in Lysozyme 2 (Lyz2) positive myeloid lineage cells by crossing Sulf1 and Sulf2 double floxed mice with the LysMcre mouse line (hereinafter LysM-Sulf-/-). At 8-12 weeks of age, the bone marrow of LysM-Sulf-/- male mice displayed myelodysplastic changes with reduced number of megakaryocytes (MK) compared to the control mice (Lyz2-cre+/-). Bone marrow-derived (BMD) macrophages from LysM-Sulf-/- mice exhibited enhanced proliferation but reduced phagocytosis of Escherichia Coli (E. Coli)in vitro. In addition, LysM-Sulf-/- BMD macrophages expressed reduced levels of key inflammatory mediators including inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α and interferon gamma-induced protein 10 (IP-10) in response to lipopolysaccharide stimulation compared to control BMD macrophages. Complete blood cell counts revealed decreases in total white blood cells (4.6 ± 0.6 10E9 Cells/L vs. 8.1 ± 1.010E9Cells/L in control mice, P = 0.0193), red blood cells (RBC, 10.9 ± 0.1 10E12 Cells/L vs. 11.4 ± 0.2 10E12 Cells/L in control mice, P = 0.0385), and hematocrit (43.5 ± 0.3% vs. 46.3 ± 0.7% in control mice, P = 0.0054) in LysM-Sulf-/- males. In addition, LysM-Sulf-/- males (8-12 weeks of age) exhibit extramedullary hematopoiesis with splenomegaly (spleen size increased by 2.6-fold, 162.1 ± 11.5 mg vs. 79.1 ± 1.2 mg in control mice, P < 0.0001). Bone and spleen morphologies are shown in the accompanied Figure. In older LysM-Sulf-/- males (8-12 months of age), the reductions in RBC and hematocrits were further exacerbated with increased reticulocyte counts in the peripheral blood. In addition, thrombocytopenia was observed in 8-12 months old LysM-Sulf-/- males (platelet counts 125.8 ± 41.4 vs. 483.3 ± 7.1 in control mice, P = 0.0007). The above changes were accompanied by amplified splenomegaly (spleen size increased by 9.2-fold, 860.4 ± 157.6 mg vs. 93.2 ± 8.2 mg in control mice, P = 0.0152) and erythropoiesis in the liver. In terms of signaling, enhanced Smad2/3 phosphorylation (primary transducers of TGF-β1 signaling) was observed in both the spleens and the lungs of LysM-Sulf-/- male mice. Finally, in contrast to the male mice, complete blood cell counts were normal in both young and old LysM-Sulf-/- females without obvious extramedullary hematopoiesis. Our investigation revealed the importance of HS 6-O-sulfation in supporting normal hematopoiesis in the bone marrow, and alterations in HS 6-O-sulfation status in the bone marrow microenvironment due to LysM-Sulfdeletion leads to not only impairment in monocyte/macrophage development and function, but also age- and sex-dependent deficiencies in erythropoiesis and thrombopoiesis. Our study identifies TGF-β1 as one of the signaling pathways regulated by the Sulfs. The regulatory roles of the Sulfs in TGF-β1 as well as other signaling pathways in hematopoiesis warrant further investigation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Therapeutic activity of GARP:TGF-β1 blockade in murine primary myelofibrosis

AbstractPrimary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by the clonal expansion of myeloid cells, notably megakaryocytes (MKs), and an aberrant cytokine production leading to bone marrow (BM) fibrosis and insufficiency. Current treatment options are limited. TGF-β1, a profibrotic and immunosuppressive cytokine, is involved in PMF pathogenesis. While all cell types secrete inactive, latent TGF-β1, only a few activate the cytokine via cell type–specific mechanisms. The cellular source of the active TGF-β1 implicated in PMF is not known. Transmembrane protein GARP binds and activates latent TGF-β1 on the surface of regulatory T lymphocytes (Tregs) and MKs or platelets. Here, we found an increased expression of GARP in the BM and spleen of mice with PMF and tested the therapeutic potential of a monoclonal antibody (mAb) that blocks TGF-β1 activation by GARP-expressing cells. GARP:TGF-β1 blockade reduced not only fibrosis but also the clonal expansion of transformed cells. Using mice carrying a genetic deletion of Garp in either Tregs or MKs, we found that the therapeutic effects of GARP:TGF-β1 blockade in PMF imply targeting GARP on Tregs. These therapeutic effects, accompanied by increased IFN-γ signals in the spleen, were lost upon CD8 T-cell depletion. Our results suggest that the selective blockade of TGF-β1 activation by GARP-expressing Tregs increases a CD8 T-cell-mediated immune reaction that limits transformed cell expansion, providing a novel approach that could be tested to treat patients with myeloproliferative neoplasms.

Treatment of congenital thrombocytopenia and decreased collagen reactivity in G6b-B–deficient mice

AbstractMice lacking the immunoreceptor tyrosine-based inhibition motif-containing co-inhibitory receptor G6b-B (Mpig6b, G6b knockout, KO) are born with a complex megakaryocyte (MK) per platelet phenotype, characterized by severe macrothrombocytopenia, expansion of the MK population, and focal myelofibrosis in the bone marrow and spleen. Platelets are almost completely devoid of the glycoprotein VI (GPVI)-FcRγ-chain collagen receptor complex, have reduced collagen integrin α2β1, elevated Syk tyrosine kinase activity, and a subset has increased surface immunoglobulins. A similar phenotype was recently reported in patients with null and loss-of-function mutations in MPIG6B. To better understand the cause and treatment of this pathology, we used pharmacological- and genetic-based approaches to rescue platelet counts and function in G6b KO mice. Intravenous immunoglobulin resulted in a transient partial recovery of platelet counts, whereas immune deficiency did not affect platelet counts or receptor expression in G6b KO mice. Syk loss-of-function (R41A) rescued macrothrombocytopenia, GPVI and α2β1 expression in G6b KO mice, whereas treatment with the Syk kinase inhibitor BI1002494 partially rescued platelet count but had no effect on GPVI and α2β1 expression or bleeding. The Src family kinase inhibitor dasatinib was not beneficial in G6b KO mice. In contrast, treatment with the thrombopoietin mimetic romiplostim rescued thrombocytopenia, GPVI expression, and platelet reactivity to collagen, suggesting that it may be a promising therapeutic option for patients lacking functional G6b-B. Intriguingly, GPVI and α2β1 expression were significantly downregulated in romiplostim-treated wild-type mice, whereas GPVI was upregulated in romiplostim-treated G6b KO mice, suggesting a cell intrinsic feedback mechanism that autoregulates platelet reactivity depending on physiological needs.

Novel insights into mouse models of ectopic proplatelet release

AbstractMature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott–Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study.

Megakaryocytes (MKs) are rare polyploid cells found in the bone marrow and produce platelets. Platelets are small cell fragments that are essential during wound healing and vascular hemostasis. In vitro differentiation of MKs from human-induced pluripotent stem cell-derived CD34+ hematopoietic stem cells (hiPSC-HSCs) could provide an alternative treatment option for thrombocytopenic patients as a platelet source. In this approach, we developed a method to produce functional MKs from hiPSC-HSCs using a xeno-free and feeder-free condition and minimize the variation and risk from animal-derived products in cell culture. We have also investigated the genome-wide expression as well as functional significance of long noncoding RNAs (lncRNAs) in hiPSC-HSC-derived MKs to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers qPCR Array Kit and identified 26 differentially regulated lncRNAs in hiPSC-HSC-derived MKs as compared with those in hiPSC-HSCs. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) was the most highly upregulated lncRNA in hiPSC-HSC-derived MKs and phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic-differentiating K562 cells. Furthermore, we have studied the potential mechanism of HOTAIRM1 based on the interactions between HOTAIRM1, p53, and miR-125b in PMA-induced K562 cells. Our results demonstrated that during MK maturation, HOTAIRM1 might be associated with the transcriptional regulation of p53 via acting as a decoy for miR-125b. Thus, the interaction between HOTAIRM1, p53, and miR-125b is likely involved in controlling cell cycling (cyclin D1), reactive oxygen species production, and apoptosis to support terminal maturation of MKs. SIGNIFICANCE STATEMENT: In vitro generation of megakaryocytes (MKs) from human-induced pluripotent stem cell-derived hematopoietic stem cells (hiPSC-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. This study has investigated the functional significance of long non-coding RNAs in hiPSC-HSC-derived MKs, which remains unclear. This study's findings suggest that the regulatory role of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in p53-mediated regulation of cyclin D1 during megakaryocytopoiesis is to promote MK maturation by decoying miR-125b.

Assessment of megakaryocytes’ size and morphology in selected myeloproliferative neoplasms

&lt;br&gt;&lt;b&gt;Introduction:&lt;/b&gt; Contemporary technological development make the cytological analysis of megakaryocytes (MKs) a valuable tool for supporting differential diagnosis of myeloproliferative neoplasms’ (MPNs) early stages.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Aim:&lt;/b&gt; The aim of the study was to identify megakaryocyte’s cytological differences between chronic myelogenous leukemia (CML), polycythemia vera (PV) and essential thrombocythemia (ET), as well as to assess the usefulness of this analysis in differential diagnosis of the aforementioned MPNs.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Materials and methods:&lt;/b&gt; Bone marrow aspirate smears were obtained from 20 patients with CML, 15 patients with PV and 12 patients with ET. The control included bone marrow biopsies from 15 patients without any hematological disorder. The following megakaryocyte’s parameters were examined: cell diameter, cytoplasmic area, nuclear area, nuclear-cytoplasmic ratio and nuclear lobulation.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Results:&lt;/b&gt; Cytological analysis revealed a significantly decreased megakaryocytes’ size and the prevalence of cells with hypolobulated nuclei in CML patients comparing to other groups. MKs in ET were found to have characteristic staghorn-like hyperlobulated nucleus, whereas polycythemia vera showed the predominance of cells with multilobulated nuclei (4-7 lobes) and a tendency to hyperlobulation. However, there were no significant differences in mean values for cytoplasmic and nuclear area among these two groups of patients.&lt;/br&gt; &lt;br&gt;&lt;b&gt;Conclusion:&lt;/b&gt; Characteristic megakaryocytes’ cytological differences in CML patients may be useful in differential diagnosis of the CML’s early stage. MKs’ size and nuclear-cytoplasmic ratio are not useful in differentiation between polycythemia vera and essential thrombocythemia.&lt;/br&gt;