Mk Development Research Articles

Serglycin controls megakaryocyte retention of platelet factor 4 and influences megakaryocyte fate in bone marrow

AbstractMegakaryocytes (MKs) produce platelets, and similar to other hematopoietic progenitors, they are involved in homeostatic aspects of their bone marrow niche. MKs release and endocytose various factors, such as platelet factor 4 (PF4)/CXCL4. Here, we show that the intra-α-granular proteoglycan, serglycin (SRGN), plays a key role in this process by retaining PF4, and perhaps other factors, during MK maturation. Immature, SRGN–/– MKs released ∼80% of their PF4, and conditioned media from these cells negatively affected wild-type MK differentiation in vitro. This was replicated in wild-type MKs by treatment with the polycation surfen, a known inhibitor of glycosaminoglycan (GAG)/protein interactions. In vivo, SRGN–/– mice had an interstitial accumulation of PF4, transforming growth factor β1, interleukin-1β, and tumor necrosis factor α in their bone marrow and increased numbers of immature MKs, consistent with their mild thrombocytopenia. SRGN–/– mice also had reduced numbers of hematopoietic stem cells and multipotent progenitors, reduced laminin, and increased collagen I deposition. These findings demonstrate that MKs depend on SRGN and its charged GAGs to balance the distribution of PF4 and perhaps other factors between their α-granules and their adjacent extracellular spaces. Disrupting this balance negatively affects MK development and bone marrow microenvironment homeostasis.

SET domain containing 2 promotes megakaryocyte polyploidization and platelet generation through methylation of α-tubulin

BackgroundMegakaryocytes (MKs) are polyploid cells responsible for producing ∼1011 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown. ObjectivesHere, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems. MethodsWe manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets. ResultsThe genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34+ cells. ConclusionThese findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

A Single-Cell Approach to Unraveling Abnormal Megakaryocyte Differentiation and Function in Myelofibrosis

Increased numbers of immature/low ploidy MKs are a hallmark feature of myelofibrosis (MF). MF MKs produce pathological amounts of pro-fibrotic factors including TGFβ causing a severe bone marrow (BM) fibrosis. Isolating MKs in MF is difficult as the fibrosis prevents BM aspiration. Also, the cellular hierarchy of normal megakaryopoiesis remains unclear, complicating any in-depth study of abnormal MK development in MF. Using a single-cell approach, we recently identified CD41 as a marker of a novel, rare population of MK-primed progenitors within peripheral blood (PB) immunophenotypic megakaryocyte-erythroid progenitors (MEP) of mobilized, healthy donor controls (Psaila et al, 2016). However, our analysis did not include MF patients and we did not analyze earlier hematopoietic stem/progenitor cells (HSPCs), which may also include MK-biased subsets that might be disrupted in MF. To examine the abnormalities in MF hematopoiesis more comprehensively and without bias from pre-selected surface markers/gene expression assays, we performed single-cell RNA-sequencing on ~50,000 individual CD34+ Lineage- PB cells of healthy mobilized donors (n=2, 18,500 cells) and 4 JAK2V617F+MF patients (n=4, 32,500 cells, 10X Genomics platform). The data were analyzed using Seurat (Sajita et al, 2015) to perform dimensionality reduction and unsupervised cell clustering and to identify differentially expressed genes. Individual cells from control and MF samples were grouped into distinct ‘clusters’ of cells with similar transcriptional profiles. Identities of the different clusters were determined by unsupervised gene ontology enrichment analyses and manual inspection of top differentially expressed genes for known lineage-specific identifiers.We observed striking differences in the cellular architecture of HSPCs in the controls and patients with MF (Fig 1A). Increased expression of MK-associated genes (incl. VWF, ITGA2B /CD41) was observed throughout HSPC populations in MF (Fig 1B). Similarly, we observed prominent clusters of CD34+ progenitors expressing PF4, a more mature MK marker, and high levels of TGFβ only in MF samples and not in controls (Fig 1B). Analyses to determine the molecular basis for the bias towards MK lineage specification in MF are now underway.As our single cell analysis showed increased expression of CD41 across a number of different HSPCs in MF patients, we compared the frequencies of CD41+, MK-primed populations in PB samples of 11 controls and 15 patients with JAK2V617F+ MF, both within more differentiated CD38+ MEP/CMP as well as primitive, CD38-neg HSPC. A selective expansion of MK-primed populations in MF was observed, with a >5-fold increase in both MK-primed MEP as well as MK-primed HSC/MPP populations, suggesting an expansion of the MK lineage and biased commitment from the apex of the HSPC hierarchy.To study more differentiated MKs, we next carried out RNA-sequencing of immature (CD41+42-) and mature (CD41+42+) MKs cultured in vitro from CD34+ HSPC from controls and patients with JAK2V617F+MF and moderate-severe BM fibrosis. The mRNA signature of immature CD42- MKs was highly distinct for MF patients vs. controls, whereas mature CD42+ MKs were not significantly different. IPA analysis identified tissue fibrosis as the most highly dysregulated pathway. The expression of genes encoding extracellular matrix proteins e.g. elastin were also markedly increased in JAK2V617F+ MKs, suggesting that MKs may themselves produce extracellular matrix factors as well as stimulating their deposition by fibroblasts. Increased expression of hepatocyte growth factor (HGF) was also observed in MF MKs. Treatment of fibroblasts in vitro with HGF or PF4 was found to inhibit TGFβ-mediated collagen deposition (P's<0.01), suggesting a complex interplay of both stimulatory (e.g. TGFβ) and inhibitory (HGF and PF4) regulators of fibrosis in MF MKs.In summary, we demonstrate that a single-cell approach enables a much finer dissection of HSPCs compared to “bulk” assays and allows proper comparison of normal and malignant cellular differentiation pathways, as well as the ability to uncover rare cell types. Our data constitute a rich and unique dataset, illustrating how transcriptomic analysis of HSPCs in MF can identify novel regulators of fibrosis with potential for therapeutic development. [Display omitted] DisclosuresMilojkovic:Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; ARIAD: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau. Mead:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau.

Human NOTCH4 is a key target of RUNX1 in megakaryocytic differentiation

AbstractMegakaryocytes (MKs) in adult marrow produce platelets that play important roles in blood coagulation and hemostasis. Monoallelic mutations of the master transcription factor gene RUNX1 lead to familial platelet disorder (FPD) characterized by defective MK and platelet development. However, the molecular mechanisms of FPD remain unclear. Previously, we generated human induced pluripotent stem cells (iPSCs) from patients with FPD containing a RUNX1 nonsense mutation. Production of MKs from the FPD-iPSCs was reduced, and targeted correction of the RUNX1 mutation restored MK production. In this study, we used isogenic pairs of FPD-iPSCs and the MK differentiation system to identify RUNX1 target genes. Using integrative genomic analysis of hematopoietic progenitor cells generated from FPD-iPSCs, and mutation-corrected isogenic controls, we identified 2 gene sets the transcription of which is either up- or downregulated by RUNX1 in mutation-corrected iPSCs. Notably, NOTCH4 expression was negatively controlled by RUNX1 via a novel regulatory DNA element within the locus, and we examined its involvement in MK generation. Specific inactivation of NOTCH4 by an improved CRISPR-Cas9 system in human iPSCs enhanced megakaryopoiesis. Moreover, small molecules known to inhibit Notch signaling promoted MK generation from both normal human iPSCs and postnatal CD34+ hematopoietic stem and progenitor cells. Our study newly identified NOTCH4 as a RUNX1 target gene and revealed a previously unappreciated role of NOTCH4 signaling in promoting human megakaryopoiesis. Our work suggests that human iPSCs with monogenic mutations have the potential to serve as an invaluable resource for discovery of novel druggable targets.

Development of platelets during steady state and inflammation.

Megakaryocytes (MK) are the sole source of platelets in the body. They develop from lineage-committed hematopoietic stem and progenitor cells (HSPCs) via intermediate cells, which differ in morphology, size, ploidy, and surface phenotype. Development and maturation of MKs is governed by different transcription factors, including GATA-1, E26 transformation-specific transcription factor (ETS) family members, nuclear factor erythroid 2 transcription factor (NF-E2), and STAT3. During such challenges as acute inflammation, platelets are consumed in high numbers and must be replenished to secure survival of the host. This is achieved by integration of inflammatory signals into early MK development and depends on the STAT1-mediated enhanced translation of transcripts in stem cell-like megakaryocyte progenitors. Here, we review recent developments, which highlight the impact of inflammation on the development of platelets from HSPCs.

Brief Report: Single-Cell Analysis Reveals Cell Division-Independent Emergence of Megakaryocytes From Phenotypic Hematopoietic Stem Cells.

Despite increasingly stringent methods to isolate hematopoietic stem cells (HSCs), considerable heterogeneity remains in terms of their long-term self-renewal and differentiation potential. Recently, the existence of long-lived, self-renewing, myeloid-restricted progenitors in the phenotypically defined HSC compartment has been revealed, but these cells remain poorly characterized. Here, we used an in vitro single-cell analysis approach to track the fate of 330 long-term HSCs (LT-HSC; Lin- cKit+ Sca-1+ CD150+ CD48- CD34-) cultured for 5 days under serum-free basal conditions. Our analysis revealed a highly heterogeneous behavior with approximately 15% of all phenotypic LT-HSCs giving rise to megakaryocytes (Mk). Surprisingly, in 65% of these cases, Mk development occurred in the absence of cell division. This observation suggests that myeloid-restricted progenitors may not derive directly from LT-HSCs but instead could share an identical cell surface marker repertoire.

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.

Recent Developments in the UNO MK 3 Process–A Low Cost, Environmentally Benign Precipitating Process for CO2 Capture

Abstract Substantial progress in the development of the UNO MK 3 process and it's cost reduction capabilities has been made since previously reported [1] . In particular, the recent results of two pilot plant trials of the process continue to demonstrate and confirm the major features of the UNO MK 3 process: Low overall cost (up to 50% less than the best amines) Multi-impurity capture and production of valuable by-products (no degradation products) Low energy of regeneration (less than 2.5 GJ/T CO 2 ) Low volatility and environmental impact (SOx and NOx converted to fertilizer products) A good fit with existing global potassium markets (no additional potassium required) These new results are groundbreaking in terms of advancing the process; with no major barriers evident for scaling up the process from the current pilot scale (TRL 5) to larger scale demonstration (TRL 6/7) over the next few years.

The Aryl Hydrocarbon Receptor (AhR) Regulates the Production of Bipotential Hematopoietic Progenitor Cells

Abstract 766The evolutionarily conserved aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix Per-ARNT-Sim family, has been studied for its role in environmental chemical-induced toxicity. Recent studies now demonstrate that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. To better understand the possible role of the AhR in hematopoiesis, we developed a novel, human, induced pluripotent stem cell (iPSC) platform based on the step-wise directed differentiation of hematopoietic progenitor cells. Our in silico analysis of transcriptional profiles of 71 primary human hematopoietic cell isolates indicated AhR-upregulation at both the hematopoietic stem cell and bi-potential megakaryocyte-erythroid progenitor (MEP) stages. This result, together with the absence of an in vitro model system enabling production of large numbers of primary human MEPs capable of differentiating into megakaryocytes (Mks) and erythroid lineage cells, motivated us to determine if AhR modulation could facilitate both MEP expansion and Mk and erythroid cell differentiation.Our results indicate that AhR has a physiological and functional role in normal hematopoietic development, and that modulation of the receptor in bi-potential hematopoietic progenitors can direct cell fate. We demonstrate a novel methodology for the directed differentiation of pluripotent stem cells in chemically defined, serum and feeder-free culture conditions into MEPs capable of final specification into Mks and/or erythroid-lineage cells. The use of a non-toxic aryl hydrocarbon receptor agonist in our directed differentiation scheme significantly increases the number of MEPs and resultant cells. Following the addition of potent AhR ligands to our cultures, we observed exponential expansion of MEPs from fifteen thousand to one billion cells in two weeks, with the role of AhR in the MEP population confirmed using a specific AhR inhibitor. This logarithmic expansion of cells appears to be a function of decreased cell death and is consistent with previous studies which suggest that the AhR can control apoptosis. In addition to allowing for the exponential expansion of MEPs, our results demonstrate that modulation of AhR can direct cell fate, with AhR agonism permissive to erythroid differentiation and antagonism favoring Mk specification. Although erythropoietin (EPO) and thrombopoietin (TPO) stimulate RBC and Mk production respectively, AhR may play a cytokine-independent role in the specification of these lineages and warrants further study.These results demonstrate a new platform for studying human red blood cell and Mk development that allows for exponentially greater production of RBCs and Mks in comparison to existing methodologies. This strategy relies on the first of its kind definition of the role of the AhR receptor in normal hematopoietic development using specialized ligands in hematopoietic progenitor cells. A useful outcome for this work will be the utilization of this in vitro platform for the clinically relevant production of blood products. An iPSC-based system, such as the one described here in which sufficient numbers of cells can be produced, should facilitate future clinical adaptation involving the transfusion of iPSC-derived red blood cells and platelets without problems related to immunogenicity, contamination, or supply. Disclosures:No relevant conflicts of interest to declare.

Inhibition of TGF-β1 signaling restores both microenvironmental and stem cells abnormalities in the Gata-1low Mouse Model of Myelofibrosis

Primary myelofibrosis (PMF) is characterized by abnormal megakaryocyte (Mk) development, fibrosis and ineffective hematopoiesis in the marrow and hematopoiesis in extramedullary sites [1]. Studies in animal models have suggested that fibrosis is established by fibroblasts activated by TGF-β1 released by the abnormal Mk. Increased levels of TGF-β1 expression in Mk have been implicated in the development of PMF. To clarify whether TGF-β1 alterations are involved in the development of PMF in Gata1low mice, the TGF-β1 content of Mk from the marrow and spleen from PMF patients and Gata1low mice was compared, the TGF-β1 pathway of the marrow and spleen of the Gata1low mouse PMF model was profiled and the consequences of pharmacological inhibition of TGF-β1 signaling, obtained through treatment with SB431542, was determined. Bone marrow (BM) sections from PMF patients contain 4-times more Mk than those from normal donors and great numbers of Mk are also detectable in their spleen. In addition, Mk from both BM and spleen of PMF patients reacted 34-times more intensely than normal Mk with the TGF-β1 antibody. Similarly the number of Mk in BM and spleen of Gata-1low mice was 2-3- fold greater than normal and these cells reacted 3-8-times more intensely with the TGF-β1 antibody than wild-type(wt)Mk. These results were confirmed by immunoelectron- microscopy. On average, one Mk from wild-type and Gata1low mice contained 10.3±2.2 and 54.3±6.5 immunogold-particles per area (p<0.01). SB431542-treatment reduced the intensity of TGF-β1 staining of Gata1low Mk both in BM (5.3±1.1) and spleen (9.2±0.7) compared to Mk both in BM (18.9±0.8) and spleen (24.3±0.9) of Gata- 1low vehicle-treated mice, while had modest effects on the expression of VEGF and CXCL12. Inhibition of TGF-β1 signaling activates hematopoiesis in BM while reducing extramedullary hematopoiesis in spleen of Gata-1low mice. In addition, it reduced fibrosis, vessel microdensity, increases Ptl counts and decreases WBC and poikilocytes in the blood of Gata1low mice suggesting a potential benefit for treatments targeting microenvironment abnormalities in PMF.

Extracellular matrix structure and nano-mechanics determine megakaryocyte function

AbstractCell interactions with matrices via specific receptors control many functions, with chemistry, physics, and membrane elasticity as fundamental elements of the processes involved. Little is known about how biochemical and biophysical processes integrate to generate force and, ultimately, to regulate hemopoiesis into the bone marrow-matrix environment. To address this hypothesis, in this work we focus on the regulation of MK development by type I collagen. By atomic force microscopy analysis, we demonstrate that the tensile strength of fibrils in type I collagen structure is a fundamental requirement to regulate cytoskeleton contractility of human MKs through the activation of integrin-α2β1–dependent Rho-ROCK pathway and MLC-2 phosphorylation. Most importantly, this mechanism seemed to mediate MK migration, fibronectin assembly, and platelet formation. On the contrary, a decrease in mechanical tension caused by N-acetylation of lysine side chains in type I collagen completely reverted these processes by preventing fibrillogenesis.

Abstract 3552: JAK/Stat3 signaling pathway modulates human megakaryocytic differentiation

Abstract Megakaryocytes (MKs) are differentiated from pluripotent hematopoietic stem cells toward a lineage capable of terminal differentiation to mature platelets. Various hematopoietic growth factors such as thrombopoietin, the ligand of c-mpl-encoded protein, have been identified as important regulators of MK development. Induction of MK differentiation may benefit disorders related to platelet dysfunction or thrombocytopenia. However, both effective inducer and clear mechanism of MK differentiation remain lacking. To clarify the regulatory mechanisms of MK differentiation, distinct inducers staurosporine (STS) and 12-O-Tetradecanoylphorbol-13-acetate (TPA) were used to assess the expression of Janus kinase (JAK) and Signal transducers and activators of transcription 3 (Stat3) signaling molecules in both human chronic myeloid leukemia K562 and erythroleukemia HEL cells. Our results demonstrated that STS and TPA effectively induced cell differentiation toward MK lineage in terms of development of polyploidy population, expression of MK-specific markers CD41 and CD61, and secretion of platelet factor 4. Intriguingly, enhancement in phosphorylation of Stat3 (p-Stat3), nuclear translocation and DNA-binding activity was evident in both STS- and TPA-treated cells. Blockade of Stat3 by Stat3 inhibitor VI and its upstream molecule JAK by JAK inhibitor I obviously declined MK differentiation. Furthermore, knockdown of Stat3 by RNA interference attenuated the expression of p-Stat3 and MK differentiation, indicating a crucial role of Stat3 in MK differentiation. In conclusion, JAK/Stat3 signaling pathway may involve MK differentiation and might be a target for development of therapeutic modulators for platelet disorders. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3552. doi:10.1158/1538-7445.AM2011-3552

Abstract 5010: Microarray analysis of gene expression profiling of essential thromocythemia (ET) and post-ET MF (myelofibrosis) derived megakaryocytic cells

Abstract Patients with ET eventually transform into Post- ET MF in most cases after several years. The underlying mechanism of the transformation is still unknown. ET and Post-ET MF is characterized by abnormal megakaryocytic proliferation. Therefore we have performed microarray analysis of gene expression profiles to compare megakaryocytes derived from patients with Post-ET MF to ET. CD 34+ cells were obtained from peripheral blood, then cultured with Serum-Free Medium of stem cell factor (100 ng/mL) and thrombopoietin (TPO 100 ng/mL), and added murine bone marrow stromal cell line OP9 as feeder layer. Megakaryocytes were then obtained by MACS selection of CD 61+ cells after 10 days culture. Then oligonucleotide microarray analysis was performed by PhalanxBio Inc. (Palo, Alto, Ca). Gene expression profiling was achieved by using the comparative cycle threshold (CT) method of relative quantification (the calibration samples were MK pools from 10 ET patients with GAPDH used as internal controls). SLR (Signal log ratio) was then calculated for comparative analysis of 2 patients with post-ET MF to a pool of ET patients. The results of post-ET MF compared with ET were as follows: 1) with analysis of 4544 genes, SLR &amp;lt;−1 were 6.58 %, and 10.12 % were &amp;gt;1, 2) in apoptosis genes, over expressed genes were IGF-1R, CFLAR, NFKBA,(GLR between 1-1.5) down-regulated genes were BAX, LGALSI, PPPIR 15A, CASP3 (GLR between -0.5 to -0.1), 3) in cell cycle controlling genes, over expressed were IL8, INSIG1, IGF-1R, BCL3(GLR between 1 to 1.5), down-regulated were PPPIR15A (GLR = -0.7). 4) in megakaryocyte-important development genes, growth factor genes including PDGF A, C were up-regulated (GLR=1.8), and VEG B was down-regulated (GLR = -1. 4):; growth factor receptor genes including IL1R 1and 2, CXCR4, IL8, PF4 were up-regulated (GLR between 1-2), IL3RA, KIT were down-regulated (GLR between -1.6 to -3); cell cycle regulator genes including CCND1, 3, RASGRP3 were up-regulated (GLR between 1 to 2.5), CDK4 was down-regulated (GLR = -1); transcription regulators including CEBPE, CEBPG were up-regulated (GLR-1.5); all Signal transducers including all STAT 1-5, MAK14 were up-regulated (GLR between 1.5 to 2.4). There are 20 up-regulated genes and 20 down-regulated genes which need further analysis for their function. These preliminary analyses of MK genes comparison between Post ET-MF and ET suggest that many anti-apoptosis, cell cycle regulator genes, growth factor genes, growth factor receptor genes and all of the signal transducer genes for the MK development genes were up-regulated while many of the pro-apoptosis genes were down-regulated. Further work to dissect the genes using the microarray analysis for the mechanism for the transformation of ET into ET-MF will be important for understanding this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2011-5010

3D Model of Thrombopoiesis.

AbstractAbstract 1609 Background.The mechanisms that regulate megakaryocytic (Mk) development within the bone marrow environment remain poorly understood. The underlying relationships between Mk maturation and bone marrow components are key factors in this process. Mk development occurs in a complex microenvironment where extracellular matrices are fundamental regulatory components. The first events occur in the osteoblastic niche and include commitment of the hemopoietic progenitor cell to Mk, arrest of proliferation and initiation of endomitosis. The second step is Mk maturation and is associated with rapid cytoplasm expansion and intense synthesis of proteins. Finally Mks, which migrate to the vascular niche, convert the bulk of their cytoplasm into multiple long processes called proplatelets that protrude through the vascular endothelium into the sinusoid lumen, where the platelets are released. Hypothesis.The hypothesis for the present work is that a complex in vitro 3D bone marrow-like environment can be used to gain fundamental mechanistic insight into cell signalling and matrix-cell interactions in the bone marrow niche related to Mk development. Methods.We propose the first 3D model for Mk function in the bone marrow environment, by refining a recently proposed bioreactor platform (Lovett et al., 2007). These bioreactors consist of 3 wells (10 mm × 15 mm × 5 mm) within a PDMS block (25 mm × 60 mm × 5 mm) which is plasma bonded to cover glass for imaging. Each bioreactor well was perfused by 23 G stainless steel needles, spanned by porous silk microtubes as blood vessel scaffolds (640 μm inner diameter), positioned approximately 500–750 μm from the bottom of the bioreactor and connected to tubing for media perfusion using a programmable syringe pump. These microtubes were prepared by dipping several times straight lengths of stainless steel wire into 10–14% (w/v) aqueous silk fibroin to obtain blood vessel scaffolds with a wall thickness of around 50 mm. Defined pore sizes of 6–8 μm were obtained by adding 6 w/t % poly(ethylene oxide) (PEO) to the silk fibroin. The perfused silk tubes comprised the vascular niche and were embedded within a cell-seeded hydrogel which comprises the osteoblastic niche. The silk microtubes were coated with a combination of fibrinogen, von Willebrand Factor, type IV collagen and SDF-1 alpha, to better establish the composition of the vascular niche. Control experiments were performed by coating silk microtubes with type I collagen. After staining human umbilical cord blood derived Mks, the cell suspension was added to the hydrogel and Mk migration was analyzed in a time-dependent manner using confocal microscopy analysis. Further, flow effluent through the vascular tubes in the bioreactor was collected at regular time intervals and platelet numbers and function were analyzed by flow cytometry and microscopy. Culture released platelets were counted as CD61+ events with the same scatter properties of human blood platelets. Results.Our results showed that Mks migrated towards the vascular microtube coated with Fibrinogen, von Willebrand Factor, type IV collagen and SDF-1. Mks were also able to complete their maturation in the proximity of the microtube by extending proplatelets. Interestingly, confocal microscopy analysis revealed that Mks were able to extend proplatelets through the vascular microtube wall and release CD61+ platelet-like particles inside the vascular microtube. Cytofluorimentric analysis demonstrated that the particles collected in the flow effluent of the vascular microtube were CD61+ cells with the same scatter properties of human peripheral blood platelets. Finally, upon coating with only type I collagen Mks did not migrate towards the vascular microtube or extend proplatelets to release platelets. Thus, by mimicking the relationship between Mks and the bone marrow environment, a model to reproduce the different steps of Mk development, such as Mk migration, proplatelet formation and platelet release, is established. This is a first significant step towards relevant systems for the study of these cellular processes in detail as well as toward potentially useful in vitro platelet production systems. Conclusions.In this work we developed a new 3D bone marrow system in vitro that could represent a new tool to understand the mechanistic basis for Mk development and function, and the diseases related to these cells. Disclosures:No relevant conflicts of interest to declare.

Study of Megakaryopoiesis and Proplatelet Formation In Myeloproliferative Disorders

AbstractAbstract 3075 Introduction.Myeloproliferative neoplsms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocytemia (ET). Several abnormalities that could explain the myeloproliferation have been reported, and most important is a striking involvement of the megakaryocyte (Mk) lineage, with hyperplasia and dysplasia resulting in an excessive production of several cytokines and chemokines. Whereas molecular defect(s) associated with the development of MPNs have been described, their pathogenesis is not thoroughly elucidated. Hypothesis.The general objective of this research was to study the process of megakaryopoiesis in MPNs in order to understand whether the pathological mechanisms underlying MPNs are intrinsic defects of Mk function or depend on an altered regulation by the bone marrow microenvironment. Patients and Methods.We analyzed in vitro the Mk differentiation and proplatelet formation in 25 PMF patients, 13 ET/PV patients, and 19 healthy controls (HCs). Mks were differentiated from peripheral blood CD34+ or CD45+ cells in the presence of TPO for 14 days. Mature Mks were, then, grown in suspension or plated onto glass coverslips coated with collagen I or fibrinogen for additional 16 hours. Mk differentiation-maturation and proplatelet formation were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Measurement of Mk diameters was performed on acquired images, at least one hundred Mks were analyzed for each sample. Mk ploidy was evaluated by flow cytometry after staining with propidium iodide. Controls were analyzed in parallel with each patient sample. Results.Mk output was higher in patients with MPNs than HCs. Patients with PMF and PV had the highest Mk output, and in PMF the number of Mks was strictly associated with the V617F JAK2 genotype. PMF Mks presented smaller size and increased nuclear/cytoplasm ratio with respect to the other MPNs and HCs. Further, the measurement of Mk diameters revealed that PMF-derived Mks rarely reached the diameter of 40 μm, while HCs and ET/PV presented larger Mks. Finally, PMF produced decreased numbers of polyploid Mks (>8N) with respect to HCs. In order to explore if defects in Mk development were associated to altered Mk function, we investigated the competence to generate proplatelet by MPN-derived and HC-derived Mks. In HC samples, a 7.5% median of the total Mks formed proplatelets, while a significant defect in extending proplatelets was observed in PMF-derived Mks with respect to other MPNs and HCs. On the contrary, patients with PV or ET showed increased numbers of Mks forming proplatelets with respect to PMF and HCs. No differences were observed between pre-fibrotic and fibrotic PMF, and pre-fibrotic PMF-derived Mks formed a decreased number of proplatelet as compared to both ET/PV and HCs. Most importantly, platelet counts correlated with Mk ability of extending proplatelets of different MPN patients. Finally, we found that the proplatelets extended by patients with PMF presented peculiar morphological features with respect to the normal counterpart and patients with PV and ET. Their more evident alteration was a significantly reduced number of bifurcations in secondary processes despite a normal length of the individual proplatelet shafts, thus indicating a defect in proplatelet branching. On the opposite, ET- and PV-derived proplatelets presented an enormous increase of bifurcations with respect to PMF and HCs. Summary and Conclusions.Mks from patients affected by MPNs presented high proliferative capacity and defects in proplatelet formation. Thus, platelet production in MPNs may not depend only on an increased number of Mks, but also on intrinsic defects in Mk function. Interestingly, our results also showed that Mk of pre-fibrotic and fully fibrotic PMF present similar biological characteristics and different from those of ET, providing a rationale for considering pre-fibrotic as a different entity from ET. In conclusion, this study proposes important new elements in the understanding of the biology of Mk and proplatelet formation in MPNs, and open a new perspective into the understanding of the pathophysiology of defects of platelet production in MPNs. Disclosures:No relevant conflicts of interest to declare.

Serum Response Factor Is An Essential Transcription Factor in Megakaryocytic Maturation.

Abstract 3652Poster Board III-588SRF is a MADS-box transcription factor first identified as an inducer of immediate-early gene expression, such as c-fos, Junb, Fosb and Egr1, in response to cytokines. It plays a critical role in cardiac and smooth muscle development and differentiation by regulating cell cycle, apoptosis, cell growth, and differentiation. SRF regulates expression of contractile and cytoskeletal genes. Its function in hematopoiesis has not yet been revealed. While no hematopoietic disease specific mutations of SRF have been identified, the region on chromosome 6 where the SRF gene is located, is a site of frequent chromosomal aberrations in MDS. MKL1, a target of the t(1;22) translocation in acute megakaryoblastic leukemia, is a cofactor for SRF mediated gene activation. In published work (Cheng EC et al., Blood 2009;113:2826), we showed that MKL1 expression increases with normal megakaryocytic (Mk) differentiation, that it promotes Mk differentiation by increasing the expression of Mk specific genes such as CD42b, and that mice lacking MKL1 have decreased platelet counts while showing increased numbers of low ploidy Mk in the BM. In addition, we showed that the effects of MKL1 on Mk differentiation require SRF. In order to test the role of SRF in Mk development, we crossed PF4-Cre mice (Tiedt R et al., Blood 2007;109:1503), which express Cre recombinase in cells committed to the Mk lineage, to SRFF/F mice (Miano JM et al., PNAS 2004;101:17132) in which functional SRF is no longer expressed after Cre-mediated excision. Our findings are quite surprising, as knockout of SRF in the Mk lineage leads to a far more profound phenotype than MKL1 KO. PF4-Cre x SRFF/F (PF4-Cre/SRF) mice are born with a normal mendelian frequency, but have significant macrothrombocytopenia. The platelet counts in WT and KO mice are 703 ± 33 × 103/μl vs 460 ± 23 × 103/μl, respectively. Despite the decreased platelet number, the BM has increased numbers and percentages of CD41+ Mk (WT: 0.41 ± 0.06% and KO: 1.92 ± 0.12%) with a predominance of Mk with hypolobated nuclei. Spleens of PF4-Cre/SRF mice show significantly increased numbers of Mk (mean of 4 and 15 Mk per high power field for WT and KO spleens, respectively). Ploidy as an assay of Mk maturation in the BM is significantly reduced in SRF −/− Mk. The percentage of CD41+ cells in SRF KO BM that is 2N is nearly 2-fold higher, and the percentage with greater than 8N ploidy is decreased by 70%. Hematopoietic stem and progenitor populations in PF4-Cre/SRF BM are unaffected, as expected due to stage and lineage specific knockout of SRF. In contrast, there is an increase in Mk progenitors in vivo in PF4-Cre/SRF mice, reflected both by FACS analysis and CFU-Mk in vitro analysis. The mechanism by which SRF disrupts Mk maturation was studied by assessment of Mk morphology, and gene expression analysis. Mk lacking SRF expression have abnormal stress fiber formation based on phalloidin staining, a phenotype parallel to that observed in mice lacking myosin heavy chain 9 (MYH9) expression, and MYH9 expression was decreased in SRF KO Mk. In summary, the data show that SRF is critical for normal Mk maturation including polyploidization and platelet production, and the mechanism by which loss of SRF disrupts megakaryocytopoiesis is, at least in part, by loss of normal expression of known targets of SRF, such as cytoskeletal genes including MYH9. Disclosures:No relevant conflicts of interest to declare.

Molecular Characteristics of Platelet Formation Studied by Genetic Profiling.

Abstract 4584Megakaryocyte (Mk) development and proplatelet formation are regulated at multiple levels although terminal stage molecular signals are incompletely delineated. We applied a customized, platelet-restricted oligonucleotide gene chip comprised of 432 genes to characterize genetic changes that occur in CD34+ cells differentiated along the Mk lineage, using a cytokine cocktail containing IL-6 (25 ng/mL), IL-11 (25 ng/mL), FLT3 (50 ng/mL), IL-1β (10 ng/mL), thrombopoietin (TPO, 50 ng/mL), and stem cell factor (SCF, 25 ng/mL). After day (D) 5, >60% of cells remained CD41 (αIIB)-positive by flow cytometric analysis, with 90% CD41-positivity at D20. Positive-selection using a β3 (glycoprotein IIIa) anti-CD61 antibody and magnetic beads was completed daily, and allowed for physical separation and comparative genetic profiles of the CD61+ and CD61- cell fractions. Gene-wise progressive correlation analysis of aggregate CD61+ to CD61- cell fractions demonstrated a correlation distribution centered between -0.2 to 0, confirming the distinct genetic profiles of CD61+ and CD61- cells. Unsupervised hierarchical clustering of the CD61+ cells – in conjunction with 41 normal platelet control profiles – confirmed the presence of two discriminatory dendograms that separated D1-15 profiles from D16-21 and normal platelet profiles, establishing that a genetic switch towards late Mk/proplatelet development occurred around D15/D16. Aggregate gene expression between D1-15 and D16-21 subsets identified 252 differentially-expressed genes (p < 0.05), only 12 of which were upregulated in D16-21 samples (11 of these 12 genes were differentially expressed between day-matched CD61- cell fractions, confirming the specificity of these changes). The conjunction of this 12-gene subset with a common 5-member pathway cluster of progressively induced genes identified three transcripts [transforming growth factor β-2 (TGFB2), CD99 antigen (CD99), and thromboxane A2 receptor (TBXA2R)] that were specifically probed to elucidate transcript and protein expression patterns in circulating platelets. Dual-color quantitative flow cytometry for these three proteins (along with ITGB3, β3, as control for a non-induced gene) was completed in both permeabilized (intracytoplasmic) and non-permeabilized (cell-surface) platelets, in parallel with thiazole orange (TO) as a measure of platelet RNA content; for all analyses, RNA and protein content in individual platelets were normalized by size and volume to adjust for size/volume differences. Despite a wide distribution of platelet size, RNA content, and protein expression, both the RNA and protein concentrations remained relatively constant. Furthermore, the RNA concentration remained tightly restricted within a narrow window, establishing that this parameter is critically maintained for all platelets unrelated to age. For all platelets, there was poor correlation (r <0.15) between RNA and protein concentrations, although correlation coefficients increased considerably in the subset of platelets with the highest (top 3%) RNA content. This was evident for both intracytoplasmic (TGFB2, r=0.41; CD99, r=0.55; TBXA2R, r=0.64) and cell-surface (TGFB2; r=0.34; CD99, r=0.73; TBXA2R, r=0.58) determinations, with smaller correlation coefficients for ITGB3 (0.21 and 0.13, respectively). In summary, these data demonstrate that (1) a genetic switch leading to mature platelet profiles (and presumably proplatelet formation) occurs at D15/D16 of MK maturation; and (2) in late-stage highly expressed transcripts, RNA concentration predicts protein concentration (both intracytoplasmic and cell-surface) in the subset of platelets with highest RNA content. These data provide insights into molecular differences between newly-formed and older platelets, and may have implications for elucidating previously-described functional differences among platelet subsets. Disclosures:No relevant conflicts of interest to declare.

Common features of megakaryocytes and hematopoietic stem cells: What's the connection?

Megakaryocytes (Mks) are rare polyploid bone marrow cells whose function is to produce blood platelets. Since the purification and cloning of the major Mk cytokine, thrombopoietin, in 1994, considerable progress has been made in understanding the biology of Mk development. Remarkably, these advances have revealed a number of key features of Mks that are shared with hematopoietic stem cells (HSCs), such as common surface receptors, lineage-specific transcription factors, and specialized signaling pathways. Why there should be such a close connection between these two cell types remains unclear. In this Prospect article, we summarize the data supporting these shared features and speculate on possible teleological bases. In particular, we focus on common links involving developmental hierarchy, endothelial cells, and bone marrow niche interactions. This discussion highlights new data showing close ontologic relationship between HSCs and specialized "hemogenic" endothelial cells during development, and functional overlap between Mks/platelets and endothelial cells. Overall, these findings may be of relevance in the development of techniques for HSC ex vivo culture and/or possible generation of HSCs via somatic cell reprogramming.

Comparative study on bone marrow megakaryocytes in children with thrombocytopenic purpura, aplastic anemia and myelodysplastic syndrome

Thrombocytopenic hemorrhage is one of the major appearance in pediatric hemorrhagic diseases, in which, idiopathic thrombocytopenic purpura (ITP) is the most common disease. Thrombocytopenia is the earliest phenomenon or the only one in certain phases of hemorrhagic diseases, such as ITP, aplastic anemia (AA) and myelodysplastic syndrome (MDS). By now, the pathogenesis of thrombocytopenia in different diseases has not been clearly determined. At present, it is very difficult to diagnose these diseases and estimate their prognosis with current clinical data. In this study, morphological characteristics and hematopoiesis function of bone marrow megakaryocyte in pediatric patients with ITP, AA and MDS were observed and the cause and mechanism of different thrombocytopenias were analyzed. There were 16 children with ITP, 17 with AA and 16 with MDS in this study. CD41 McAb immunohistochemical technique was used to detect micromegakaryocyte on bone marrow smears. Plasma clot culture and CD41 McAb immunohistochemical technique were used for the MK-colony forming assay. The colony formation rate of colony formation unit-megakaryocyte (CFU-MK) and burst formation unit-megakaryocyte (BFU-MK) were counted. There was no statistical difference on the positive rates of micromegakaryocyte and type I lymphoid small micromegakaryocyte between groups of ITP and control. The number of micromegakaryocyte and the formation rates of CFU-MK in ITP group were significantly higher than those in control group. Among AA patients, the numbers of MK, micromegakaryocyte and the formation rates of CFU-MK, BFU-MK in vitro significantly decreased. There was no significant difference in the positive rate of micromegakaryocyte between groups of MDS and control, but the number of micromegakaryocyte and the positive rate of type I lymphoid micromegakaryocyte were significantly higher than those of control group. There was no statistical difference of the formation rate of CFU-MK between groups of MDS and control. But in 63% childhood patients, the formation rate of CFU-MK decreased, 25% increased,and 13% was normal; BFU-MK formation rate decreased significantly in MDS group. Overproliferation of bone MKs may exist in most ITP patients. For obviating the nosogenetic factors, the normal MK releasing platelet could be easily found in the culture system. But the colony formation rate of MK decreased in a few patients with CITP. The abnormality of MK might be one of the reasons for thrombocytopenia in partial patients with ITP. Underproliferation of MKs may exist in AA, but no pathosishemogenesis was found. The dysfunction of early phase MK progenitor and stem cell might be the major reason for AA, but not the abnormality of hematopoietic microenvironment. There may be two kinds of megakaryocyte clones in bone marrow of children with MDS. One may be pathologic and potentially malignant micromegakaryocytes, the other may be the normal megakaryocytic precursors. The increase of pathologic MK resulted in abnormal development and maturation of MK in bone marrow. The change of megakaryopoiesis showed different in ITP, AA or MDS. Using bone marrow smear megakaryocyte counting, small micromegakaryocyte immunohistochemical detecting and the formation rate of bone marrow MK colony assay, the different thrombocytopenia can be diagnosed during the early stage of ITP, AA or MDS.