MK Cell Research Articles

Megakaryocytes Have Immune Characteristics in Murine Immune Bone Marrow Failure

Introduction Megakaryocytes (MKs) have familiar roles in the bone marrow (BM) microenvironment, including platelet production, maintenance of hematopoietic stem and progenitor cells (HSPCs) function, and more recent evidence suggests MK engagement in immune responses. Immune aplastic anemia (AA) is characterized by oligoclonal expansion of T-cells and death of HSPCs leading to bone marrow failure (BMF). As MKs demonstrate niche maintenance and immune effector function, we explored the potential roles of MKs in the development of AA in a murine model of immune BMF. Methods BMF was induced in CByB6F1 mice by intravenous infusion of lymph node (LN) cells from major histocompatibility complex mismatched C57BL/B6 (B6) donor mice at a dose of 30-40x10 6 cells/recipient. No irradiation was given to recipients to avoid extra damage to MKs and CByB6F1 recipient mice were sacrificed at D7, D10, D14, and D17 following LN cell infusion (Chen J, et al. Blood. 2004). Peripheral blood (PB) samples, tibias, femurs, and sterna were harvested at each timepoint. Immunophenotyping of MKs and T cells was performed by flow cytometry on flushed BM cells and two-photon excitation microscopy (2P) imaging was performed on fixed sternal samples, using fluorescence-labelled CD41 antibody to visualize the physical location of MKs in BM. To enrich for MKs, BM cells were filtered through a 10 µm pluriStrainer™ through which most lymphocytes, red blood cells, and other small cells were eluted while MKs and other large size cells remained on the strainer. Large cells and residual small cells were subsequently assessed by single cell RNA sequencing (sc-RNAseq) on the Parse Bio Evercode™ platform based on combinatorial barcoding technology, which was able to accommodate the large size of MKs, as no partitioning step is needed in the analysis workflow. Results BMF was successfully induced as measured by decreasing PB counts from point of lymphocyte infusion accompanied by increasing expansion of CD4 and CD8 T-cells and marked reduction of total BM cells. MKs persisted throughout the course of BMF as measured by both flow cytometry and 2P (Figure 1A) despite PB thrombocytopenia and progressive marrow hypocellularity. Concomitantly, MKs identified by large size on forward scatter versus side scatter and CD41+ expression had progressive upregulation of immune effector markers IA-IE and CD53 over the course of BMF. Following sc-RNAseq and bioinformatics analysis of enriched BM cells at D14, cell clustering identified a distinct cell cluster representing MKs (Figure 1B). MK identity was supported by co-expression of transcripts encoding platelet factor 4, TUBB1, Glycoprotein 5, and Itga2b (CD41). This MK cell population was distinct from macrophages which highly expressed Fcgr4, Cd5l, Ccr5, Cd74 and Gbp5 genes. Gene expression profiling demonstrated that MKs in BMF mice showed upregulation of immune pathways such as interferon gamma and interferon alpha response and downregulation of pathways related to platelet activation and platelet homeostasis, as compared with normal mice. Conclusions In a murine model of BMF, using multiple modalities, we demonstrate that MKs cease platelet production, manifested by reduction in peripheral blood platelet count and downregulation of gene expression pathways related to platelet production, while concurrently acquiring characteristics of immune effector cells. This suggests that MKs are active participants in immune BMF.

Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity

AbstractMegakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished β1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.

Trisomy 21-Associated Abnormalities in IGF Signalling and the Fetal Microenvironment Both Contribute to Disruption of Fetal Hematopoiesis in Down Syndrome

In Down syndrome (DS), trisomy 21 (T21) causes perturbation of fetal liver (FL) hematopoiesis leading to expansion of megakaryocyte-erythroid (MK-E) progenitors. On this background, FL hematopoietic stem/progenitor cells (HSPC) may acquire GATA1 gene mutations which usually present as a neonatal preleukemic condition, Transient Myeloproliferative Disorder (TMD). TMD develops only in fetal life and most cases spontaneously regress after birth, suggesting unique features of the T21 fetal microenvironment may be important in driving both abnormal DS FL hematopoiesis and mutant GATA1 clones in TMD. Previous work showed activation of insulin-like growth factor (IGF) signalling in TMD and DS-AMKL and fetal-specific IGF dependence of murine MK development implicating T21 in altered fetal IGF signalling.To investigate the role of IGF signalling in fetal MK-E development in DS FL without GATA1 mutations, we performed gene expression profiling (GEP) of DS FL (n=5) and normal FL CD34+ cells (n=3). GSEA showed significant enrichment of IGF targets. We therefore measured expression of the principal IGF receptor, IGF1R, on DS FL HSPC (n=6). Surface IGF1R was uniformly expressed on all DS FL HSPC subpopulations. However, there was no difference in IGF1R surface or gene expression between DS and normal FL HSPC (n=8) suggesting that increased IGF1R expression on TMD and DS-AMKL cells is an early event and may reflect their fetal rather than T21 origin. To determine if the enrichment of IGF targets in DS FL HSPC might reflect increased IGF production in the FL microenvironment, we compared IGF expression in DS FL mesenchymal stromal cells (MSC)(n=4) and hepatocytes with normal FL MSC (n=4). Both DS and normal FL MSC expressed large amounts of IGF2, but little or no IGF1 with no difference between DS and normal MSC. Similarly, IGF2 was expressed by IHC at similar levels in both DS and normal fetal hepatocytes and MSC whereas IGF1 was barely detectable. Normal and DS fetal bone marrow (BM) MSC also expressed high levels of IGF2, but not IGF1 in contrast to adult BM MSC. Thus, IGF2 is the main IGF produced in the human fetal hematopoietic environment. Consistent with a specific role in DS FL hematopoiesis, IGF2 caused marked proliferation of DS FL clonogenic MK and MK-E cells (n=3) and stimulated MK proliferation in liquid culture while in normal FL, IGF2 stimulated EPO-independent BFU-E and Pre-BFU-E but had no effect on MK cells. Since differential effects of IGF2 on DS/normal FL HSPC might be due to altered expression of the Insulin Receptor (InsR), which binds IGF2 with low affinity, we compared InsR surface and gene expression in DS and normal FL HSPC but found no significant difference. IGF2 also binds to IGF2R, a negative regulator of IGF2, however there was no difference in IGF2R expression between DS and normal FL HSPC. Co-culture of normal FL CD34+ cells with DS or normal FL MSC (n=3) promoted MK and erythroid cell proliferation and differentiation. Since the effect of DS FL MSC on normal FL CD34+ cells was significantly greater (~4-fold) than that of normal FL MSC, the data above suggest this is unlikely to be due to IGF2 alone. To investigate whether T21 alters the MSC secretome, we performed GEP of DS FL MSC (n=3) and compared this with normal FL MSC (n=5). Both normal FL and DS FL expressed known hematopoietic cytokine genes, including SCF, CXCL12, G-CSF, M-CSF, Flt3 and IL-6, as well as IGF2 but not IGF1, EPO or TPO. Of the top 80 differentially expressed genes (Fold Change>2), ~40% encode secreted proteins, including several which affect IGF availability.Conclusion: T21 perturbs FL hematopoiesis through both HSPC-intrinsic mechanisms, including changes in IGF responsiveness, and through alterations to the FL microenvironment. DisclosuresNo relevant conflicts of interest to declare.

The Effect of the R882H DNMT3a Mutation on Megakaryocyte Cell Lines

IntroductionDNA methyltransferase (DNMT) family play an important role in the development and growth of lives, encoding enzymes that catalyze the addition of a methyl group to the cytosine residue of CpG islands. With the increase in methylation, the downstream genes are often associated with reduced expression. In this family, DNMT3a occupies the essential position to implement the de novo methylation. Timothy J. Ley and many other scientists found that in M4 and M5 acute myeloid leukemia (AML), around 20% patients suffered from DNMT3a mutation (most are R882H mutation), always associating with adverse prognosis. But what’s the reason for adverse prognosis? Additionally, our formal Meta analysis showed that the de novo AML patients with DNMT3a mutation have higher platelet counts, WBC and RBC counts. To shed some light on the possible causal relation between the increasing in platelet count and poor prognosis led by DNMT3a mutation, we transduced the MK cell lines with genes null-mCherry (null), DNMT3a wild type-mCherry (DNMT3aWT) and DNMT3a R882H mutation type-mCherry (DNMT3aMT) respectively, trying to figure out the possible role that the mutation plays in the megakaryopoiesis and thrombopoiesis. Also, we tested several drugs that may target the mutation.MethodsThe SFFV-null-IRES-mCherry, SFFV-DNMT3aWT-IRES-mCherry and SFFV-DNMT3aMT-IRES-mCherry plasmids were constructed by Dr. Qianli Jiang, modified from LEGO-iC plasmids. MK cell lines (chrf-288-11, meg-01) were flow-through transduced with the lentivirus produced by packaging plasmids and those above. All the fluorescence positive cells have been doubly sorted by flow cytometry. Cell ploidy was analyzed by flow cytometry using Propidium Iodide (PI); colony forming unit (CFU-MK) were enumerated 14d after being plated with TPO and IL-3; cell proliferation were tested by CCK-8; apoptosis was measured via flow cytometry with PI and Annexin V-FITC; CD41a and CD61 were tested with flow cytometry. The drug tests including Decitabine, Dasatinib and Rituximab were analyzed using CCK-8 test and cytomorphologic tests.ResultsWith CCK8 test of chrf-288-11 and meg-01, DNMT3aMT proliferates faster than the null and DNMT3aWT (P<0.05, Fig.1). In CFU-MK, both cells lines showed that DNMT3a mutation promoted the colony formation (P<0.05). The CD41a percentage decreased from null to DNMT3aWT and DNMT3aMT (P<0.05) while the CD61percentage increased from null to DNMT3aWT and DNMT3aMT (P<0.05). Also, with morphologic analyses, DNMT3aMT in both cell lines maintain more mature stages. Cell ploidy test also demonstrated that cell lines with DNMT3a mutation contain more multiploids (P<0.05). Apoptosis test illustrated that DNMT3a mutation protect the cell lines from apoptosis (P<0.05). In the drug experiments, 1uM Decitabine could slow down the proliferation of 3 gene types of chrf-288-11 significantly (P<0.05). Dasatinib also posed a negative effect on the proliferation of 3 gene types of chrf-288-11 (P<0.05, Fig.2). In Rituximab experiment, we could find that interestingly, certain concentrations could speed up the proliferation of 3 gene types of chrf-288-11, while others not (P<0.05, Fig.3).ConclusionWith all the above evidences, we can safely conclude that the megakaryocyte cell lines with DNMT3a mutation are associated with high-differentiation, high-colony formation and low-apoptosis, which could help us to understand the elevation of platelet count in AML patients with DNMT3a mutation. The anti-apoptosis and renewal ability of the cell lines with DNMT3a mutation may lead to a bad prognosis of these AML patients (with DNMT3a mutation). What’s more, according to the drug experiment, we found in the both cell lines, DNMT3aWT and DNMT3aMT cells died significantly at even low concentration of decitabine. Dasatinib also slowed down the proliferation of 3 gene types of chrf-288-11, whether Dasatinib could lead to further treatment of such leukemia with DNMT3A mutation needs more research. Rituximab is helpful in the treatment against refractory thrombocytopenia. However, the mechanism hasn’t been clarified. Interestingly, our results showed that, Rituximab may have a direct effect on MKs, giving a boost to the megakaryopoiesis and thrombopoiesis with certain concentration. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Megakaryocytes are essential for HSC quiescence through the production of thrombopoietin

Tissue homeostasis demands regulatory feedback, suggesting that hematopoietic stem cell (HSC) activity is controlled in part by HSC progeny. Yet, cell extrinsic HSC regulation has been well characterized only in niche cells of non-hematopoietic origin. Here we identify feedback regulation of HSCs by megakaryocytes (Mks), which are mature hematopoietic cells, through production of thrombopoietin (Thpo), a cytokine pertinent for HSC maintenance. Induced ablation of Mk cell population in mice perturbed quiescent HSCs in bone marrow (BM). The ablation of Mks resulted in decreased intra-BM Thpo concentration presumably due to Thpo production by Mks. Thpo administration Mk ablated mice restored HSC functions. Overall, our study establishes Mk as an essential cellular component of the HSC niche and delineates cytokine-oriented regulation of HSCs by their own progeny.

Alternative Methods Of Thrombocytopenia: Patients With ITP Have Increased Megakaryocyte/T Cell Interactions In The Bone Marrow and Higher Serum TRAIL Levels

AbstractThe pathology of immune thrombocytopenia (ITP) is not fully understood. Although antiplatelet antibodies (APAs) are thought to be causative, these are not detected in up to 30% of patients. In addition, T cell directed therapy is frequently successful. Patients with ITP display cytotoxic activity towards platelets, and abnormalities in T cell repertoire. However, there is incomplete understanding of the interactions between T cells and megakaryocytes within the bone marrow. We hypothesized that if T cell or NK cell cytotoxicity against megakaryocytes contributes to thrombocytopenia, then T and/or NK cells should be in direct contact with the megakaryocyte in the bone marrow. We therefore further explored the interactions between T cells and megakaryocytes in bone marrow trephine biopsies - taken at or near to diagnosis - in patients with ITP. To further explore apoptotic pathways, serum TRAIL levels were also assessed at different time points in disease. MethodsBone marrow trephine samples were taken as clinically indicated at or around the time of diagnosis and processed as per department protocol. Samples from patients with known ITP (n=9) were further processed and stained with CD3, CD8, CD4 and CD56. Immunohistochemistry was performed using Leica Bond-max automated immunohistochemistry system. The density of MK cells, CD3, CD8 and CD4 was recorded and the numbers of MK in contact with T or NK cells were recorded on multiple sites on the trephine. Control bone marrows from staging marrows for lymphoma were used for comparison (n=6). For further analysis of the non-antibody component of ITP, serum TRAIL levels were measured in 63 samples from 43 patients at different stages of disease. Patients had all consented for research. ResultsResults of the bone marrow examination are shown in the table. Bone marrow samples from patients with ITP had higher density of megakaryocytes than control bone marrows (median 41/mm3 compared to 20/mm3 - p=0.03). Megakaryocytes appeared normal in morphology. CD3 density was also higher in patients with ITP, although this was not significant (p=0.17). There was however increased interaction between CD3 and MK cells in patients with ITP compared to controls (median 50% of MK cells in contact with CD3 cells in ITP compared to 32% in controls - p=0.02) – figure 1a. There was no correlation between MK cell density, T cell density and MK-T cell interaction, making this finding unlikely to have occurred by chance. In ITP patient samples further analysis of T cell type was undertaken and shown in the table showing interactions occur between both CD4s and CD8s and the MK cells (figure 1b). Four patients had reverse CD4:8 ratios in the bone marrow. Interaction between MK and CD56+ cells were less frequent (0 to 8%). There was no association with response to treatment and MK or T cell findings, although patients who responded to rituximab appeared to have lower MK density. Patients who responded to thrombopoietic (TPO) agonists - and by virtue of their use, had not responded to other treatments – had a high density of MK cells at diagnosis (bone marrows were taken at diagnosis, and before TPO agonists were started). Serum TRAIL levels were higher in patients with chronic ITP and with platelets <50 when compared to patients in remission (platelets > 150) (p<0.1). In patients with serial samples, there was an inverse relationship between TRAIL and platelet count.ITP PatientsControlsMK Density (per mm2)41.2 [13.2 – 76.7]19.6 [10.8 – 33.3]CD3 Density (per mm2)511.8 [297 – 805.9]317.5 [209 – 609]T-Cell (CD3)/MK Cell Interaction (%)50 [29 – 75]32 [17 – 41]CD4/MK Cell Interaction (%)52 [25 – 71]N/ACD8/MK Cell Interaction (%)25 [13 – 33]N/ANK Cell/MK Cell Interaction (%)6 [0 – 8]N/A ConclusionPreliminary evidence suggests increased interactions between T cells and megakaryocytes in the bone marrow of patients with ITP. The demonstration of interactions between CD3 cells and MK cells in both patients and controls shows the importance of the MK cell in the immune system. Further investigation is under way to explore whether CD8 or NK cell interaction is only found in patients with ITP, and whether these interactions result in MK cell death, or reduction in platelet production. High TRAIL levels in patients with chronic disease, refractory to first and second line therapy further suggests that increased apoptosis plays an important role in the ongoing thrombocytopenia in chronic disease. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Serotonin and Its Metabolism Enhances Megakaryopoiesis and Proplatelet Formation

Serotonin (5-HT) has been recently identified as a novel growth factor. We previously demonstrated that 5-HT enhances murine megakaryopoiesis via 5-HT2 receptors and has promotor effect on hematopoiesis(Yang M et al, Stem Cells, 2007). However, the molecular mechanism remains under explored. In the terminal stage of mammalian megakaryocyte development, platelets are released from proplatelet protruding from megakaryocytes via cytoskeleton reorganization. 5-HT is shown to modulate cell migration and remolding by activating cytoskeleton reorganization, but the effects of 5-HT on proplatelet formation have not been investigated. Our results showed that 5-HT significantly promoted human CFU-MK formation and reduced apoptosis on human megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5HT2 receptor inhibitor. 5-HT also stimulated proplatelet formation through activating the p-Erk1/2 expression and F-actin reorganization. Melatonin, the metabolism of 5-HT, promoted the recovery of platelets and the formation of bone marrow colony forming units in irradiated mice. Our findings suggested that 5-HT and melatonin plays an important role in human megakaryopoiesis. Interaction of 5-HT and 5-HTR2B induced downstream activation of PI3-k/Akt signal pathway leading to human MK cell proliferation. In addition, activation of 5-HTR2B also induced Erk1/2 phosphorylation, which then promoted cytoskeleton reorganization and subsequent proplatelet formation. We also proved that melatonin exerts a protective effect on MK and platelets in the irradiation mice model. Disclosures: No relevant conflicts of interest to declare.

Profiling Deacetylase Activities in Cell Lysates with Peptide Arrays and SAMDI Mass Spectrometry

The development of arrays that can profile molecular activities in cells is important to understanding signaling pathways in normal and pathological settings. While oligonucleotide arrays are now routinely used to profile global gene expression, there is still a lack of tools for profiling enzyme activities in cell lysates. This paper describes the combination of peptide arrays formed on self-assembled monolayers and mass spectrometry to provide a label-free approach for identifying patterns of enzyme activities in cell lysates. The approach is demonstrated by profiling lysine deacetylase (KDAC) activities in cell lysates of the CHRF megakaryocytic (Mk) cell line. Class-specific deacetylase inhibitors were used to show that terminal Mk differentiation of CHRF cells is marked by a pronounced decrease in sirtuin activity and by little change in activity of KDACs 1-11. This work establishes a platform that can be used to identify changes in global activity profiles of cell lysates for a wide variety of enzymatic activities.

Shear-Optimized Platelet-Like-Particles From High Ploidy Mks: From Segregation to Composition and Activation

Abstract 3456Megakaryocytes (MKs) are rare cells that generate about 1010 platelets every day, which are necessary for vascular homeostasis via clot formation and contraction. Understanding MK differentiation and platelet production is of broad clinical importance and extends to a need to augment platelet numbers in patients. Our group has demonstrated that reversible but sustained inhibition of non-muscle myosin-II (NMM-II) increases MK polyploidization, proplatelet formation, and membrane flexibility, thereby increasing platelet generation (Shin, Spinler, et al., PNAS, 2011; 108:11458–63). The terminal steps of hematopoiesis involve the coordinated maturation and migration of multipotent hematopoietic stem cells (HSCs) from the bone marrow to the perivascular niche. Bulky, polyploid MKs do not easily transmigrate into blood, but do extend tubular membrane ‘proplatelet' projections into the bloodstream. Visualization by others has shown that blood shear stress fragments the projections into circulating platelets, motivating a controlled study of shear effects on MKs in vitro.To recapitulate this environment in vitro, a cone and plate rheometer is used to impart physiologically relevant shear stress on MEG01s, a human MK cell line. These experiments reveal that platelet-like-particle (PLP) generation is sensitive to both biomechanical and pharmacological factors namely blebbistatin inhibition of NMM-II. We demonstrate that shear stress reduces phosphor-deactivation of NMM-II heavy chain at Ser1943 to approximately 30% of the unsheared level, which indicates a restoration of NMM-II activity necessary for proper platelet function. Stimulation of rheometer generated PLP cultures with collagen-I showed aggregation and phosphatidylserine exposure (with Annexin-V binding in the presence of Ca2+). These data demonstrates that PLPs generated in this system retain some degree of functionality such that MKs exposed to shear stress and blebbistatin result in approximately 6.5 fold more PLPs than untreated MK cultures.Other groups have shown the transport and assembly of platelet organelles to be microtubule dependent and occur de novo along proplatelets (Italiano, et al, Blood, 2005; 106:4066–75). To assess partitioning and segregation of proteins from MKs in sheared membranes, we used fluorescence-imaged micro-deformation (FIMD) to monitor CD41 and NMM-IIA during micropipette aspiration. Studies of erythrocytes had already shown a rich variety of membrane component responses to membrane distension (Discher, et al, Science, 1994; 266:1032–5), but the methods have not yet been applied to MKs. Antibody labeling of MK surface CD41 shows a homogeneous intensity along the aspirated projection of membrane, but cell body shows approximately 3 fold higher intensity, suggesting an excess of CD41 in the MK. Pre-treatment with blebbistatin increases fragmentation frequency, and these fragments show a similar trend with CD41 expression. Nucleofection was used to introduce either GFP tagged WT NMM-IIA or phosphomemetic, myosin deactivating, GFP tagged NMM-IIA S1943D to assess whether pSer impacts partitioning of this cytoskeletal protein that is abundant in platelets. Both WT and S1943D NMM-IIA are seen in the aspirated cell projection, but WT NMM-IIA clearly accumulates at the leading edge of the aspirated projection and at sites of membrane fission and fragmentation, whereas S1943D remains uniformly dispersed. These findings thus underscore the central role that NMM-II heavy chain phosphorylation, and thus activity, in proplatelet formation and platelet fragmentation. Disclosures:No relevant conflicts of interest to declare.

Umbilical Cord Blood-Derived Stromal Cells Regulate Megakaryocytic Proliferation and Migration Through SDF-1/PECAM-1 Pathway

We have previously reported that human umbilical cord blood-derived stromal cells (hUCBDSCs) are able to enhance the expansion of CFU-Meg in vitro, particularly promote the megakaryocytic lineage recovery, and effectively protect the survival of irradiated mice. In this study, we demonstrated that hUCBDSCs secreted SDF-1 to stimulate PECAM-1 expression in HEL cells (MK cell line), and consequently promoted the proliferation and migration of HEL cells. On the other hand, SDF-1 knock down in hUCBDSCs or PECAM-1 knock down in HEL cells diminished or abrogated the above effect. In addition, SDF-1/PECAM-1 probably activated PI3K/Akt and MAPK/ERK1/2 pathways. This report for the first time defines a SDF-1/PECAM-1 signaling pathway in the proliferation and migration of MKs, which provides supportive evidence for the clinical applications of hUCBDSCs in the treatment of megakaryocytic injury.

The Importance of Matrix Elasticity and Shear Force on Megakaryocytic Differentiation

Abstract 1329 Introduction.Environmental (matrix) elasticity (Engler et al., Cell 126: 677 (2006)) and biomechanical forces (Baruch et al., Blood 114:1975 (2009)) are important differentiation signals for stem cells. In this study, we investigated how these biomechanical/biophysical signals affect megakaryocytic (Mk) differentiation, aiming to uncover signaling mechanisms behind these effects, and establish better culture environment for Mk differentiation and platelet biogenesis. CHRF cell (Fuhrken et al., Exp. Hematol. 35: 476 (2006)), a well- established Mk cell line, was used in this study. Upon PMA stimulation, CHRF cells undergo Mk-like differentiation, including endomitosis and projection of cytoplasmic extensions. Methods and Results.Application of shear stress (2.5 dyne/cm2; physiological range: 1.3∼4.1 dyne/cm2) to CHRF cells attached to fibronectin-coated surfaces for two hours resulted in retraction, shedding and formation of cytoplasmic extensions (proplatelet-like structures), whereas there were no morphological changes in static culture. Shear force also accelerated endomitosis as assessed by flow-cytometry based analysis of BrdU incorporation: CHRF cells exposed for two hours to shear flow had 16% more BrdU positive cells compared to cells under static conditions. The stress responsive p53 is a regulator in both cell-cycle arrest by laminar flow (Zeng et al., JBC 278: 24594 (2003)) and Mk differentiation (Fuhrken et al., JBC 283: 15589 (2008)). To determine if p53 is involved in the response of CHRF cells to shear stress, western analysis was used to show that two hours of exposure to shear (2.5 dyne/cm2) increased the ratio of K382-acetylated p53 to total p53 by more than 188% (n=3, p<0.05) while total p53 protein expression did not change significantly. These data suggest that p53 acetylation is mediating, partially at least, the response of Mk cells to biomechanical forces.In order to examine the effect of matrix elasticity on Mk differentiation, polyacrylamide gels were used to control elasticity. We coated the gel surface with fibronectin to provide holding points for CHRF cells to sense gel elasticity. As matrix elasticity increased, the % of CHRF cells with cytoplasmic extensions increased first and decreased later (Fig. 1, n=3, Error bar: SEM). The highest percentage (17%) of CHRF cells displaying cytoplasmic extensions was observed on a gel with elasticity (ca. 8.2 kPa) mimicking the in vivo vascular niche. Non-muscle myosin IIA (NMMIIA) has been shown to serve as elasticity sensor in mesenchymal stem cells. Thus, we aimed to investigate the role of NMMIIA in the response of CHRF cells to matrix elasticity. Blebbistatin, an inhibitor of NMMIIA, minimized the differences of the percentage of cells with cytoplasmic extensions between gels with different elasticity (Fig. 2), thus suggesting that NMMIIA is involved in the response of CHRF cells to matrix elasticity and may also serve as a biological elasticity sensor. As shown by fluorescent microscopy, the cellular distribution of NMMIIA was directed by matrix elasticity (Fig. 3). On soft gels (75 Pa; n=3), NMMIIA was concentrated in the center of the cytoplasm. On a moderately stiff gel (5.6 kPa; n=3), NMMIIA was distributed across the whole cell, while on glass (the stiffest condition tested; n=3), NMMIIA moved into the nucleus and the tips of cytoplasmic extensions. Others have shown NMMIIA is involved in regulation of gene expression when it moves into the nucleus, so our observations that NMMIIA translocates into the nucleus as gel elasticity increases strongly suggests that NMMIIA may regulate gene transcription during proplatelet formation and platelet biogenesis. In future we want to investigate and prove this hypothesis. [Display omitted] Conclusions and Significance.Our data shed light on the largely unexplored role of biomechanical and biophysical parameters (of physiological significance) on Mk differentiation. The impact of these parameters is both of physiological and practical significance in the context of platelet biogenesis and ex vivo platelet generation. Disclosures:No relevant conflicts of interest to declare.

Role of tumor suppressor p53 in megakaryopoiesis and platelet function

The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on invivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets invitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our invivo and exvivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.

Covalently immobilized glycosaminoglycans enhance megakaryocyte progenitor expansion and platelet release

Application of umbilical cord blood (UCB) transplantation in adults as a treatment post-chemotherapy is hampered due to delayed platelet recovery. A potential solution suggested is the transfusion of ex vivo expanded megakaryocytes (Mks) from hematopoietic stem cells (HSCs). Alternatively, large-scale production of platelets in vitro has also been attempted with the goal of transfusing them into patients with thrombocytopenia. Glycosaminoglycans (GAGs) have been shown to influence the proliferation and differentiation of HSCs. This study sought to examine the effects of immobilized GAGs on the expansion, apoptosis, and platelet release activity of CD41a+ Mk progenitors in vitro. Freshly isolated HSCs from UCB were cultured in serum-free media supplemented with thrombopoietin on GAG-derivatized chitosan membranes for 17 days. Controls consisted of uncoated and chitosan-coated wells. Wells were demidepopulated at periodic intervals and analyzed by flow cytometry. Heparin and dermatan sulfate surfaces significantly enhanced total cell and Mk cell expansion (p < 0.05) compared to both the controls. The apoptotic Mk fraction was significantly lower on GAG surfaces (p < 0.05) compared to the polystyrene control during the early stages of the culture (days 7 and 11). However, by day 17, the apoptotic Mk fraction was comparable on all surfaces. The cumulative number of platelets generated on dermatan sulfate and heparan sulfate surfaces was significantly higher (p < 0.05) than on both the controls. These results suggest that immobilized GAGs delay Mk apoptosis and thereby enhance Mk expansion and platelet production.

Characterization of the Impact of Culture on the Thrombopoietic Potential of Cord Blood Progenitors.

AbstractAbstract 3710Co-transplantation of ex vivo expanded progenitors with unmanipulated hematopoietic stem cells improves the recovery of neutrophils while that of platelets remains problematic especially for cord blood (CB) transplantation. The lack of efficacy on platelet recovery could be the result of poor expansion of megakaryocyte progenitors (Mk-P) and Mks induced by the cytokine cocktails currently used, or to the lack of optimization of this procedure. Therefore, we recently developed two cytokine cocktails by statistical design of experiment, OMPC and BS1 for the expansion of CB Mk-P and for the differentiation of CD34+ cells into Mk, respectively. Our objectives were now to compare the short term thrombopoietic potential of CB cells expanded with these cocktails to that of unexpanded cells, and to investigate the impact of two culture parameters, the temperature of incubation and the length of expansion, on platelets recovery.CB CD34+ cells were expanded in serum-free medium with the cocktail BS1 (SCF, TPO, IL-6, IL-9) or OMPC (SCF, FL, TPO). The total progeny of 130,000 expanded CD34+ cells were transplanted into irradiated NOD/SCID mice (9 mice/group). Unexpanded CD34+ cells were transplanted as control to determine whether this procedure procures any advantages to platelet recovery. Human platelets (hPLT) were monitored in the peripheral blood by cytometry, as well as human bone marrow (BM) engraftment.Since we previously showed that Mk expansion and Mk purity can be improved by incubating CB cultures at 39°C, we first assessed the impact of mild hyperthermia (MH) on the thrombopoietic potential of OMPC-expanded cells. hPLTs levels 4 days post-transplant (PT) achieved with cells expanded at 37°C were low and insignificant vs the PBS-control group (p>0.05). In contrast, hPLT levels were 20-fold greater in mice transplanted with cells cultured at 39°C (median of 0.7 vs 13.7 hPLT/uL, respectively p<0.002). No differences were then observed up to 36 days-PT, at which time hPLT levels became greater (8.5-fold) in mice injected with cells expanded at 37°C (p<0.04). Next, the short-term hPLT potentials of CB cells expanded for 6 days were compared to that of unmanipulated CD34+ cells. Only low and insignificant hPLT levels (p>0.05 vs PBS-control group) were detected 4- and 7 days-PT with the unexpanded cells (median < 1.4 hPLT/uL). In contrast, hPLT were readily detected in 94±8% of mice injected with OMPC- and BS1-expanded cells (9- and 5-fold greater at day 4, and 26- and 32-fold greater at day 7, respectively; p<0.05 vs unexpanded and PBS controls). Conversely, hPLT levels in mice injected with unexpanded cells became and remained greater by 11 days PT (p<0.05). Finally, we investigated the impact of the length of expansion (6, 10 and 14-days) on the thrombopoietic potential of expanded CB cells. Mice transplanted with day-6, day-10 and day-14 BS1-expanded cells received an average dose of 1.3, 11.3 and 22.7 millions CD41+ Mks. Despite the increasing Mk cell doses, an inverse correlation between the length of expansion and hPLT levels were observed 14- (p<0.001) and 28-days PT (p<0.005). A similar correlation was observed for the number of huCD41+ cells in the BM 14-days PT (p<0.01). An inverse correlation between the length of culture and the total number of CD34+ and GPA+ human BM cells was also evident. As previously observed, hPLTs levels 14-days PT were the highest in mice transplanted with the unexpanded cells (p<0.002). High hPLT levels were detected in all cohorts up to 16 weeks PT, with the highest level observed in the unmanipulated and day-10 expanded groups.In summary, hPLT levels derived from unmanipulated CB cells were insignificant prior to 11 days PT, while hPLTs were readily detected with OMPC- and BS1- expanded CB cells. However, hPLTs production from unmanipulated cells became and remained dominant by 11–14 days PT. These results also confirm that cytokine cocktails promoting Mk-P expansion can improve short term platelet recovery and that expansion under MH is of added benefit for short term hPLT recovery.In conclusion, this work reveals that the hPLT reconstitution kinetics differ considerably between expanded and unmanipulated CB cells. Importantly, this difference support the concept that co-transplantation of both cell source could be of significant benefit for the short term platelet recovery in the context of CB transplantation. We are now in the process of evaluating this potential synergy. Disclosures:No relevant conflicts of interest to declare.

Targeting Non-Histone Protein Acetylation Impairs Platelet Production During Normal Megakaryocytopoiesis.

AbstractAbstract 2610Megakaryocytopoiesis consists of a succession of events in which MK progenitors initially proliferate and acquire lineage-specific markers, followed by polyploidization and cytoplasmic maturation. MK maturation culminates in the formation of cytoplasmic extensions (i.e. proplatelets) that leads to platelet shedding into the circulation. Panobinostat (LBH589) is a histone deacetylase inhibitor that has antiproliferative and cytotoxic effects on several types of cancer cells including blood cells from patients with hematological malignancies. One of the major adverse events associated with LBH589 treatment is thrombocytopenia. In this study, we hypothesize that the effects of LBH589 on thrombopoiesis might occur by targeting acetylation of histone and/or non-histone proteins resulting in defective platelet production. To test this hypothesis we investigated the effects of LBH589 on megakaryocytopoiesis in MK cell lines (i.e. HEL JAK2V617F positive cells) and in primary human MK. First, we tested the effects of LBH589 on the ability of human CD34+ cells to generate MK colony forming units (CFU-MKs). Neither CFU-MK or CFU-MIX derived colony formation was reduced in the presence of LBH589. To evaluate the effects of LBH589 on parameters of MK maturation, MK were generated in vitro from peripheral blood-derived CD34+ cells by employing an expansion culture system containing SCF and TPO for 6 days followed by 8 additional days incubation in the presence of TPO. These studies were pursued in the presence or absence of LBH589. Treatment with LBH589 did not significantly influence the number of CD61+ MK (i.e. control = 55.8%; 2.5nM LBH589 = 45.2%, p value=0.109; 5nM LBH589=38.5%, p value=0.095, of viable 7-AAD−/CD61+ cells) or the degree of polyploidization (i.e. control = 17.4%; 2.5nM LBH589 = 14.4%86.7, p value=0.157; 5nM LBH 589=12.8%, p value=0.116, cells with >4N DNA content). Culture-derived platelets were analyzed phenotypically and quantitated by means of dual labeling with anti-CD41 antibodies and with thiazole orange (TO) in order to identify new reticulated platelets. The percentage of CD41+/TO+ platelets derived from MK generated in the presence of LBH589 was significantly reduced (i.e. 2.5nM LBH589=11%, p value 0.046 and 5nM LBH589=9%, p value=0.011, CD41+/TO+ cells) as compared with MK generated in the absence of LBH589 (18.5% CD41+/TO+ cells). These findings were consistent with the observation of significant numbers of proplatelet-bearing MKs in control cultures but not in LBH 589-treated cultures. Collectively, these data suggest that LBH589 impairs platelet production while having a minimal effect on MK commitment, cytoplasmic maturation or polyploidization. To better understand the mechanisms responsible for such effects on thrombopoiesis, RNA extracted from control MK and from MK treated in vitro with LBH589 was analyzed by real time quantitative PCR to evaluate GATA-1 and NF-E2 expression. GATA-1 and NF-E2 mRNA levels were unchanged after treatment with LBH589. We found, however, that LBH589 induced a 4.8 to 7.5-fold increase in histone H3 acetylation. These data suggest that the negative impact of LBH589 on MK maturation was not mediated by its effects on chromatin but rather was possibly due to its effects on acetylation of nonhistone proteins. We demonstrated that LBH589 treatment increased acetylation of tubulin, a non-histone cytoplasmic protein that is a component of the microtubule (MT) cytoskeleton. The later stages of MK maturation are highly dependent on MT which represent the structural scaffold for proplatelet extension and enables the transport of cytoplasmic organelles into nascent platelets. The changes in the acetylation status of tubulin are critical for proper MT function and are mediated by HDAC6 which we found by Western blot analysis to be inhibited by LBH589 treatment. Based on these findings we suggest that LBH589-induced changes in tubulin acetylation result in aberrant MT function which in turn, leads to defective proplatelet and platelet formation. These nonhistone protein modifications might serve as a drug target for the development of novel agents (LBH589) to treat patients with extreme thrombocytosis due to underlying myeloproliferative neoplasms. Disclosures:Iancu-Rubin:Novartis: Research Funding. Hoffman:Novartis: Research Funding.

Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Abstract 2531Poster Board II-508 BACKGROUND AND HYPOTHESIS.We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS.8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS.At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS.Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures:No relevant conflicts of interest to declare.

The Effect and Underlying Mechanism of Melatonin on Platelet Formation and Survival in a Thrombocytopenic Model.

Thrombocytopenia is a major treatment-related problem for cancer patients on chemo- or radiotherapy. In clinical studies, melatonin has been shown to be able to lower the frequency of thrombocytopenia in these cancer patients. However, the actual effects and underlying mechanism of melatonin on megakaryocytopoiesis, and platelet formation/survival have not been formally investigated. Our previous studies have demonstrated that serotonin can stimulate hematopoiesis and megakaryocytopoiesis (Yang et al, Stem Cells, 2007). Melatonin is an acetylated product of serotonin. To delineate the effect of melatonin on platelet production, we hypothesize that melatonin has therapeutic effects on thrombocytopenia by promoting megakaryocytopoiesis and enhancing the recovery of platelets via activation of Akt/Erk signaling. The effect of melatonin on thrombopoiesis was investigated in an irradiated mouse model (4 Gy). Eighteen mice were randomly divided into three groups (6 in each). Group 1 (normal control, N) received no irradiation or melatonin; Group 2 (model control, C) and Group 3 (melatonin, M) received 4 Gy total body irradiation. After irradiation, melatonin (10mg/kg.d) was injected intraperitoneally into Group M for consecutively 21 days. Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from the three groups on day 0, 7, 14, and 21.Our results showed that melatonin enhanced the recovery of platelets and WBC counts. We also investigated the in-vitro effect of melatonin on CFU-MK formation at different doses (0–-500 nM). The results showed that melatonin significantly promoted CFU-MK formation at a dose dependant manner. The maximal concentration was at 200 nM (P<0.01, n=6). The size of CFU-MK with melatonin treatment was much larger and each MK cell was more mature. Melatonin also stimulated the formation of bone marrow CFU-F. Using megakaryocytic cell CHRF-281, we further found that melatonin exerted protective effect on serum-free induced apoptosis of CHRF cells. The cell viability was significantly increased with the treatment of melatonin. Flow cytometric dot-plot analysis demonstrated that the population of apoptotic cells (Annexin V/PI staining) was significantly decreased with melatonin treatment (200nmol/L) for 72 hrs (n=4). It also showed trends of amelioration of caspase-3 expression and the proportion of cells containing JC-1 monomers (indicating a trend in the drop of mitochondrial membrane potential). The population of phospho-Akt and Erk1/2 were significantly increased after treatment by melatonin. Our results showed that melatonin had a promoting effect on platelet formation and survival in a thrombocytopenic model. The underlying mechanisms may be involved in directly stimulating megakaryocytopoiesis and having anti-apoptotic effect in megakaryocytes via the activation of Akt/Erk signaling.

Glycoprotein Ibα Promoter Drives Megakaryocytic Lineage-Restricted Expression After Hematopoietic Stem Cell Transduction Using a Self-Inactivating Lentiviral Vector

Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34(+) cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbalpha, GpIIb, or EF1alpha gene regulatory sequences. Both GpIbalpha and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbalpha promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbalpha gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34(+) hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes

Megakaryocytic (Mk) cell maturation involves polyploidisation, and the number of platelets produced increases with Mk DNA content. Ploidy levels in cultured human MK cells are much lower than those observed in vivo. This study demonstrated that adding the water-soluble vitamin nicotinamide (NIC) to mobilised peripheral blood CD34+ cells cultured with thrombopoietin (Tpo) more than doubled the percentage of high-ploidy (> or = 8N) MK cells. This was observed regardless of donor-dependent differences in Mk differentiation. Furthermore, MK cells in cultures with NIC were larger, had more highly lobated nuclei, reached a maximum DNA content of 64N (vs. 16N with Tpo alone), and exhibited more frequent and more elaborate cytoplasmic extensions. NIC also increased the ploidy of cultured primary murine MK cells and a cell line model (CHRF-288) of Mk differentiation. However, NIC did not alter Mk commitment, apoptosis, or the time at which endomitosis was initiated. Despite the dramatic phenotypic differences observed with NIC addition, gene expression microarray analysis revealed similar overall transcriptional patterns in primary human Mk cultures with or without NIC, indicating that NIC did not disrupt the normal Mk transcriptional program. Elucidating the mechanisms by which NIC increases Mk maturation could lead to advances in the treatment of Mk and platelet disorders.

Microarray Functional Comparison of CD34+ and Megakaryocytic Cell Transcriptomes in Myeloid Metaplasia with Myelofibrosis.

Myeloid Metaplasia with Myelofibrosis (MMM) is a myeloproliferative disorder (MPD) associating ineffective and extramedullary hematopoiesis with progressive splenomegaly, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ cells with the prominent amplification of dystrophic megakaryocytes (Mk). We compared the transcriptome of CD34+ and Mk cell from the peripheral blood (PB) of MMM patients and from the PB and bone-marrow (BM) of unmobilized healthy donors. Application of multivariate analyses (principal component analysis and classification by partitioning around medoids algorithm) on Gene Ontology annotation of differential genes allowed a global functionally approach of the main cellular dysregulated pathways in MMM. Each sample cRNA probe was individually and differentially hybridized to the cRNA reference probe on a Human Oligo-microarray 22K (Agilent) and data were normalized by application of the local LOWESS algorithm. Comparison of the CD34+ cell transcriptome between MMM and healthy donors revealed a global down regulation of 2/3 of the expressed genes in contrast to 1/3 of genes that are up-regulated after data filtration by significance analysis microarray (SAM) algorithm (threshold p<0.01). The down-regulation mainly concerns genes involved in hematopoiesis inhibition such as interferon pathway and of lymphoid and apoptotic functions such as BCL6. Such a downregulation is in favour of a lost of survival/proliferation suppression signals rather than of a positive activation process. Up regulated genes included genes linked to Mk differentiation/function, hemostasis and migration process such as tetraspanines and chemokines. In contrast to CD34+ cell, comparison of the transcriptomes from MMM and control Mk revealed an approximate equal number of down- and up-regulated genes after data filtration by Student test algorithm at p-value threshold p<0.05. The gene expression profile of MMM Mk cell exhibited an intense metabolic disturbance (16S proteasome), infidelity of DNA repair compensated by an increase in the control of DNA damage (RINT-1) and possible disturbance of the angiogenic message as show by the down regulation of the TNNI3 troponin. Modulation of these genes might participate in the dysmegakaryopoiesis and the angiogenesis that features MMM. Comparison of CD34+ and Mk cell transcriptomes from patients and healthy donors shows that the two populations exhibit distinct expression profiles. No common differential genes that could be a unique signature of MMM have been identified, confirming the multifactorial and multistep feature of this heterogenous disease. Our data are in agreement with the main pathological features of MMM, especially concerning key genes involved in cell cycle status, proliferation, mobilization and transduction pathways. Furthermore, among these differentially expressed genes, the CD9 tetraspanine and FLT3 genes could be individualized and might represent molecular markers for the diagnosis of JAK2 V617F negative MMM and to distinguish MMM from other MPD.