Human Megakaryocytes Research Articles

Imetelstat (GRN163L), a Telomerase Inhibitor Selectively Affects Malignant Megakaryopoiesis in Myeloproliferative Neoplasms (MPN)

Imetelstat is a telomerase inhibitor which has been shown to have therapeutic activity in patients with myelofibrosis (MF) (Tefferi A. et al., 2013 ASH Annual Meeting, Abstract 662) and essential thrombocythemia (ET) (Baerlocher G. et al., 2012 ASH Annual Meeting, Abstract 179). In clinical trials involving patients with a variety of other malignancies, the primary dose-limiting toxicity of GRN163L has been thrombocytopenia. We utilized GRN163L in order to explore the possible role of telomerase on human megakaryocytes (MK) and the mechanisms underlying the drug’s inhibitory effects on platelet production. MK were generated from normal primary CD34+ cells using an ex vivo culture system previously described by our laboratory (Iancu-Rubin C. et al, Blood 2011, 117:17; 4580-4589). We first showed that both telomerase activity and the expression of its catalytic unit hTERT were elevated in proliferating normal CD34+ cells, declined transiently during the initial stages of MK differentiation but were then partially restored during the final stages of terminal maturation. Treatment of normal CD34+ cells with GRN163L did not affect the numbers of CFU-MK assayed. Furthermore, exposure to GRN163L during the first 7 days in a liquid culture system did not interfere with the ability of CD34+ cells to commit to MK. When the same cultures were allowed to mature for 7 additional days, the proportion of CD34+/CD41+ MK precursors in drug-treated cultures was twice that observed in control and the drug-treated cultures contained 70% fewer mature CD41+/CD42b+MK than control cultures. The inhibitory effects of GRN163L on MK maturation were supported by observations showing that the cultures treated with the drug contained 60% fewer polyploid MK than control cultures. These results suggest that that GRN163L-mediated inhibition of telomerase does not affect normal MK progenitors but blocks the maturation of MK precursor cells.Previous studies have shown that GRN163L inhibits neoplastic CFU-MK colony formation by CD34+ cells from patients with ET (Brunold C. et al., 2011 ASH Annual Meeting, Abstract 3843). We, therefore, examined the effects of GRN163L on malignant MK from patients with MF and ET. Unlike normal CD34+ cells, treatment of MPN PB-MNCs with GRN163L decreased the numbers of assayable CFU-MK from 6 out of 11 patients. CFU-MK colony formation by PB-MNCs from these 6 patients was reduced by 33% (ranging from 13% to 50% reduction in CFU-MK formation) as compared to PB-MNCs treated with an inactive form of the drug (p value= 0.00473). Of note, in two out of 5 patients in which the total number of CFU-MK was not affected by GRN163L treatment, the drug decreased the size of the CFU-MK colonies formed. The ability of MPN PB-MNCs to differentiate into MK was next assessed. Although the total number of cells in PB-MNC liquid cultures exposed to GRN163L was decreased as compared to those treated with the inactive drug (n=6; p value=0.03204), the proportion of CD34+/CD41+MK precursors generated was not significantly affected. We then evaluated the effects of GRN163L on the genotype of MPN MK generated in the presence and absence of GRN163L by assessing the JAK2V617F allele burden. Treatment with GRN163L but not the inactive form of the drug reduced the JAK2V617F allele burden in MK derived from two patients: in one patient, JAK2V617F allele burden in MK generated in the presence of the inactive drug was 91.86% while MK generated in the presence of GRN163L was 19.75%; MK generated from a second patient had a JAK2V617F allele burden of 10.84% in the presence of the inactive drug which was reduced to 2.14% in the presence of GRN163L.We conclude that GRN163L-mediated inhibition of telomerase affects normal megakaryopoiesis by blocking the terminal maturation of CD34+/CD41+ MK precursors, providing a possible explanation for GRN163L’s propensity to induce thrombocytopenia in patients with normal bone marrow. By contrast, GRN163L treatment inhibited the ability of MPN CD34+ cells but not normal CD34+ cells to form CFU-MK colonies and drug treatment reduced the numbers of malignant MK generated. We propose that the amelioration of fibrosis observed in a clinical trial of MF patients treated with GRN163L might be due to, at least in part, two potential modes of action: 1) inhibiting malignant MK progenitors cells and 2) preventing terminal MK maturation thus depleting the pool of mature MKs which are the major source of fibrogenic cytokines in MF. DisclosuresIancu-Rubin:Geron Corporation: Research Funding. Parker:Geron Corporation: Employment. Eng:Geron Corporation: Employment. Hoffman:Geron Corporation: Consultancy.

Ex Vivo Induction of Megakaryocytic Differentiation of Umbilical Cord Blood CD34+ Cells

Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34+ cells.Design and Methods: Firstly, we expanded CD34+ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34+ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×107 cells from the second stage of culture.Results: After the first stage culture, proliferation folds of total cells and CD34+ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34+ cell was between 1.0×104 to 1.5×104 CD41+ megakaryocytes with a purity of CD41+ and CD42+ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes).Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future. DisclosuresQin:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Identification of the Human Unipotent Megakaryocyte Progenitor, and Its Pathophysiological Roles in Human Thrombopoietic Disorders

▪In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP; CD34+ CD38+ IL3Ra- CD45RA-), which resides downstream of common myeloid progenitor (CMP; CD34+ CD38+ IL3Ra+ CD45RA-). However, the definition of unipotent Meg progenitor (MegP) is still controversial and, in the hierarchical map, where Meg potential mainly resides remains unclear. Here, we report prospective isolation of unipotent MegP, which is endowed with much more robust Meg potential than MEP, and emerges directly from CMP bypassing MEP.Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (7.9 ± 3.2 %) of CMPs expressed CD41, but none of the other CD34+ subpopulation including hematopoietic stem cells (HSCs; CD34+ CD38- CD45RA-), MEPs and granulocyte-macrophage progenitors (GMPs; CD34+ CD38+ IL3Ra+ CD45RA+) expressed significant levels of CD41. Purified CD41+ CMPs retained myeloblast-like immature morphological characteristics with large and round nucleus and basophilic cytoplasm, closely resembling those of CD41- CMPs and more immature HSCs. Interestingly, cells with immature 4N-nuclei were exclusively found in CD41+ CMPs (about 1%), indicating that a minor fraction of this population is initiating endomitosis and polyploidization process, an important characteristic of megakaryoblasts. A conventional In vitro culture condition with fetal bovine serum and a myeloerythroid cytokine cocktail revealed that CD41+ CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41- CMPs generated all types of myeloid colonies. These unexpected observations led us to hypothesize that CD41+ CMPs were committed to the Meg lineage. We then cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. In this condition, the majority of single CD41+ CMPs gave rise to pure megakaryocyte colonies, without forming any other myeloerythroid colonies. In addition, in cDNA microarray analysis, CD41+ CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulation of erythroid and GM lineage-affiliated genes, reflecting their lineage potential. Therefore, we defined the CD41+ CMP as a human MegP.The Meg colony-producing potential of MegPs was almost thirty­­ times as strong as that of MEPs on per-cell-basis (672 ± 232 vs. 20 ± 6.2 colonies per 1000 cells) and MegP-derived Meg colonies accounted for more than ninety percent of those from whole CD34+ CD38+ progenitor populations, indicating that this population is the major source of megakaryocytes and platelets production rather than MEP. The MegP population was found in 7 day culture of CD41- CMPs but not of MEPs, suggesting that MegPs are downstream of CMPs, but are independent of MEP. More importantly, MegP pool dramatically expanded in essential thrombocythemia (ET) patients’ bone marrow, constituting three times larger part of CMP than that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We are currently further analyzing ET patients’ bone marrow to obtain data regarding V617F JAK2 mutation frequency, for example. Thus, the newly-identified MegP robustly contributes to physiological and pathological human megakaryopoiesis, thus could be a therapeutic target in various thrombopoietic disorders. DisclosuresNo relevant conflicts of interest to declare.

EZH2 Mutations Are Drivers of Clonal Hematopoiesis and Leukemic Transformation in a Mouse Model of Primary Myelofibrosis

Primary Myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by aberrant myeloid differentiation, associated with disruption of the bone marrow niche with subsequent fibrosis development and a high risk of leukemic transformation. The phenotypical complexity observed in PMF likely reflects the heterogeneous mutation profile of the neoplastic stem cells driving the disease. In our former work, we identified a CD133+ hematopoietic stem / progenitor cell (HSPC) population from patient peripheral blood that can drive major PMF morbidity parameters in a xenotransplantation mouse model. Mutational analysis of the JAK2 locus at the single cell level within the CD133+ population showed highly variable levels of cells with a JAK2+/+, JAK2V617F/+, or JAK2V617F/V617F genotype, indicating that clonality is unlikely driven by JAK2 mutations. In two of these patient samples, and in a third patient sample with CALR-fs* mutations, we identified a high load of missense mutations in EZH2 (45 to 95%), suggesting they may be critical for the clonal expansion of the neoplastic stem cell compartment.EZH2 mutations are found in circa 7% of PMF patients and are correlated with poor prognosis. EZH2 is a critical enzymatic subunit of the Polycomb Repressor Complex 2, which initiates gene repression of select genes through its intrinsic activity for methylating lysine-27 of histone H3 (H3K27). To date, the exact contribution of EZH2 mutations to PMF evolution or AML transition has not been clarified. CD133+ HSPC carrying EZH2 mutations either with JAK2 or CALR mutations were transplanted into immunodeficient NOD-scid-gamma (NSG) mice. Mice engrafted with patient samples carrying either EZH2-Y633C and JAK2-V617F or EZH2-Y733* and CALR-fs* mutations showed a strikingly similar phenotype, including high human cell engraftment (10-20%), skewed myelopoiesis, dysplastic human megakaryocytes, splenomegaly, anemia, and fibrosis in either the BM or spleen. In the case of xenotransplanted mice receiving CD133+ cells with a low JAK2 burden and EZH2-D265H mutations, we observed the highest engraftment in our mouse model (62-95%) and in one case AML transition with >50% CD133+ human blasts in murine bone marrow. Notably, AML arose from a CD133+ EZH2D265H/+ cell that lacked JAK2V617Fmutation.We thus conclude that EZH2 mutations confer to CD133+ neoplastic stem cells a predisposition to clonal aberrant hematopoiesis; whereas acquisition of JAK2V617F or CALR mutations likely leads to the observed myeloproliferation and disruption of megakaryocytic and erythroid regulation . Moreover, our results demonstrate that epigenetic mutations (like EZH2D265) and not JAK2V617F are critical for AML transition. Our data underscore the importance of post-transcriptional modifiers of histones in altering the epigenetic landscape of neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones. DisclosuresNo relevant conflicts of interest to declare.

ARHGEF12 Is Essential for Human Megakaryocyte Differentiation and Plays Critical Roles in Platelet Function

Megakaryocytopoiesis, the process by which hematopoietic stem cells develop into mature megakaryocytes (MK), and thrombopoiesis, platelet production/release, are critical for blood homeostasis. We tested the hypothesis that the Rho guanine exchange factor, ARHGEF12 (also known as LARG), is critical for MK differentiation and platelet functions based on the following: 1) ARHGEF12 is part of a recurrent translocation with MLL in acute myeloid leukemia. 2) Both published microarray datasets and deep-sequencing data from our lab on primary human CD34+ cells differentiating into MKs show that ARHGEF12 expression goes up dramatically during MK differentiation. 3) ARHGEF12 is one of the most highly expressed guanine exchange factors in platelets. 4) ARHGEF12 forms a complex with G proteins and stimulates Rho-dependent signals. It is known that platelet activation can be initiated by extracellular stimuli working through G protein-coupled receptors and Rho signaling, suggesting that ARHGEF12 may function in platelet activation. 5) Mice with KO of RhoA (a known ARHGEF12 substrate) in the MK-lineage have macrothrombocytopenia and defective platelet activation.To test this hypothesis, we used ARHGEF12 shRNA mediated KD and an ARHGEF12 specific pharmacological inhibitor (Y16) in both murine and human primary cells, and characterized a LARG KO mouse model for MK and platelet phenotypes, and found:ARHGEF12 is differentially upregulated during MK differentiation and is enriched in plateletsUsing quantitative RT-PCR and western blot analysis at different timepoints of primary FACSorted Mk progenitors induced to differentiate into mature MK in vitro, ARHGEF12 RNA and protein expression increases during MK differentiation in both the murine and human systems. Also western blot analysis of murine platelet rich plasma shows that ARHGEF12 protein is highly expressed in platelets.ARHGEF12 is essential for human MK differentiationTo test the function of ARHGEF12 in Mk differentiation, we used lentiviral shRNA to knockdown ARHGEF12 in FACSorted primary human Mk progenitors from mobilized peripheral blood differentiated in vitro to MK. The results show that ARHGEF12 knockdown blocks MK polyploidization (not shown) and maturation (Fig. A). This was confirmed using a published ARHGEF12 inhibitor (Y16) in the differentiation culture of human MK progenitors, in which there was a dose-dependent block in MK differentiation (Fig. B). These data suggested that ARHGEF12 is essential for human MK differentiation.We researched the function of ARHGEF12 in the murine system using a constitutive ARHGEF12 knockout mouse model. The mice have enlarged platelets (p=0.07) and a decreased platelet count (p=0.01). However, the knockout mice have normal BM cellularity with no change in megakaryocyte number or ploidy, suggesting that ARHGEF12 is dispensable for murine MK differentiation in vivo.ARHGEF12 is essential for platelet function in both the murine and human systems:To test whether ARGEF12 functions in platelet activation, we compared WT versus KO platelet activation in vitro. We tested activation in response to ADP, U46619 (Thromboxane), ADP+U46619, and Thrombin. KO plateelts have significantly reduced activation in response to U46619 and thrombin, with no effects on ADP-induced activation. Analogous studies using the ARHGEF12 inhibitor (Y16) on WT platelets revealed supportive evidence. Lastly, we tested ARHGEF12 function in human platelet aggregation using the Y16 compound. Consistent with the murine data, Y16 blocked platelet aggregation in response to both U46619 and Thrombin. Taken together, these data strongly suggest that ARHGEF12 is essential for platelet function and acts downstream of the Thromboxane and Thrombin receptors.In summary, we found that ARHGEF12 is differentially up-regulated in MK differentiation both in human and in mouse system,. It plays a critical role in human Mk differentiation but is dispensable in murine MK differentiation, and ARHGEF12 is critical for platelet functions in both human and mouse systems, potentially acting downstream of Thromboxane and Thrombin receptors. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human megakaryocyte progenitors derived from hematopoietic stem cells of normal individuals are MHC class II-expressing professional APC that enhance Th17 and Th1/Th17 responses

Platelets, like stromal cells, present antigen only via MHC class I, but the immune potential of their progenitors has not been explored in humans. We derived CD34+CD117+CD41+CD151+ megakaryocyte progenitors (MKp) in vitro from mobilized peripheral blood hematopoietic stem and progenitor cells (HSPC) of normal subjects using culture conditions akin to bone marrow niche, or organs that support extramedullary hematopoiesis. The MKp expressed MHC Class II in contrast to platelets and functioned as professional APC before they matured further. Moreover, MKp constitutively expressed mRNA encoding mediators for human Th17 expansion, including IL-1, IL-18, IL-6, TGFβ, IL-23, BAFF, and COX2. MKp also expressed high levels of type I interferon and IRF5 mRNA. In contrast to platelets, MKp augmented the expansion of Th17, Th1, and potent Th17/Th1 double-positive cells in normal PBMC and CD4 line T cells from normal subjects or lupus patients. The Th cell augmentation involved pre-committed memory cells, and was significant although modest, because only non-cognate MKp-T cell interactions could be studied, under non-polarizing conditions. Importantly, the MKp-mediated expansion was observed in the presence or absence of direct MKp-T cell contact. Furthermore, MKp augmented Th17 responses against Candida albicans, a serious opportunistic pathogen. These results indicate an immunologic role of MKp in situations associated with extramedullary hematopoiesis and mobilization of HSPC.

Aspirin influences megakaryocytic gene expression leading to up-regulation of multidrug resistance protein-4 in human platelets.

AimThe aim of the study was to investigate whether human megakaryocytic cells have an adaptive response to aspirin treatment, leading to an enhancement of multidrug resistance protein-4 (MRP4) expression in circulating platelets responsible for a reduced aspirin action. We recently found that platelet MRP4 overexpression has a role in reducing aspirin action in patients after by-pass surgery. Aspirin enhances MRP4-mRNA levels in rat liver and drug administration transcriptionally regulates MRP4 gene expression through peroxisome proliferator-activated receptor-α (PPARα).MethodsThe effects induced by aspirin or PPARα agonist (WY14643) on MRP4 modulation were evaluated in vitro in a human megakaryoblastic DAMI cell line, in megakaryocytes (MKs) and in platelets obtained from human haematopoietic progenitor cell (HPC) cultures, and in vivo platelets obtained from aspirin treated healthy volunteers (HV).ResultsIn DAMI cells, aspirin and WY14643 treatment induced a significant increase in MRP4 and PPARα expression. In human MKs grown in the presence of either aspirin or WY14643, MRP4 and PPARα-mRNA were higher than in control cultures and derived platelets showed an enhancement in MRP4 protein expression. The ability of aspirin to modulate MRP4 expression in MKs and to transfer it to platelets was also confirmed in vivo. In fact, we found the highest MRP4 mRNA and protein expression in platelets obtained from HV after 15 days' aspirin treatment.ConclusionsThe present study provides evidence, for the first time, that aspirin treatment affects the platelet protein pattern through MK genomic modulation. This work represents an innovative and attractive approach, useful both to identify patients less sensitive to aspirin and to improve pharmacological treatment in cardiovascular high-risk patients.

Human megakaryocyte progenitor emerges independent of megakaryocyte/erythrocyte progenitor

Human megakaryocyte progenitor emerges independent of megakaryocyte/erythrocyte progenitor

Proteasome function is required for platelet production.

The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.

HGH promotes megakaryocyte differentiation and exerts a complementary effect with c-Mpl ligands on thrombopoiesis

AbstractHuman growth hormone (hGH) is known to play a functional role in regulating hematopoiesis, although its direct effect on thrombopoiesis is unclear. In this study, we show for the first time that hGH has a distinct capacity to promote the differentiation of human primary megakaryocytes derived from umbilical cord blood CD34+ cells. In particular, hGH is potent in facilitating proplatelet formation and platelet production from cultured megakaryocytes. The stage- and time-specific activations of extracellular signal-regulated kinase 1/2 and protein kinase B signaling pathways are involved in the action of hGH. Fusion with hGH enhances the effect of a tandem dimer of thrombopoietin mimetic peptide (dTMP) on thrombopoiesis, manifested by a significant acceleration and increase of platelet production, indicating that hGH may exert a complementary and synergistic effect with c-Mpl ligands on thrombopoiesis. Accordingly, the administration of dTMP-growth hormone fusion protein led to a rapid platelet recovery in mice with severe thrombocytopenia induced by 6.5 Gy total body irradiation, thereby markedly abridging the duration of thrombocytopenia crisis (platelets <150 × 109/L), in comparison with high doses of dTMP. These findings demonstrate the functional role of growth hormone in promoting thrombopoiesis and provide a promising avenue for the treatment of severe thrombocytopenia.

Novel function of the chromosome 7 open reading frame 41 gene to promote leukemic megakaryocyte differentiation by modulating TPA-induced signaling

12-O-tetradecanoylphorbol-13-acetate (TPA) activates multiple signaling pathways, alters gene expression and causes leukemic cell differentiation. How TPA-induced genes contribute to leukemic cell differentiation remains elusive. We noticed that chromosome 7 open reading frame 41 (C7ORF41) was a TPA-responsive gene and its upregulation concurred with human megakaryocyte differentiation. In K562 cells, ectopic expression of C7ORF41 significantly increased CD61 expression, enhanced ERK and JNK signaling, and upregulated RUNX1 and FLI1, whereas C7ORF41 knockdown caused an opposite phenotype. These observations suggest that C7ORF41 may promote megakaryocyte differentiation partially through modulating ERK and JNK signaling that leads to upregulation of RUNX1 and FLI1. In supporting this, C7ORF41 overexpression rescued megakaryocyte differentiation blocked by ERK inhibition while JNK inhibition abrogated the upregulation of FLI1 by C7ORF41. Furthermore, we found that Y34F mutant C7ORF41 inhibited megakaryocyte differentiation. nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was the major activator of C7ORF41 that in turn repressed NF-κB activity by inhibiting its phosphorylation at serine 536, while MAPK/ERK was the potent repressor of C7ORF41. Finally, we showed that C7ORF41 knockdown in mouse fetal liver cells impaired megakaryocyte differentiation. Taken together, we have identified the function of a novel gene C7ORF41 that forms interplaying regulatory network in TPA-induced signaling and promotes leukemic and normal megakaryocyte differentiation.

Milestones in understanding platelet production: a historical overview

The discovery of thrombopoietin (TPO, also termed THPO) in 1994 was a major achievement in understanding the regulation of platelet production. In prior decades, physiological studies had demonstrated that platelets were produced from bone marrow megakaryocytes and that the megakaryocytes responded to thrombocytopenia by increasing their number, size and DNA ploidy. In 1958, it was proposed that a 'thrombopoietin' must exist that regulated this interaction between the circulating platelet mass and the bone marrow megakaryocytes. After over three decades of effort, TPO was finally purified by five independent laboratories. TPO stimulated megakaryocyte colony-forming cell growth and increased the number, size and ploidy of megakaryocytes. When the genes for TPO or TPO receptor were eliminated in mice, megakaryocytes grew and platelets were made, but at 15% of their normal number. A first generation of recombinant human (rh) TPO molecules [rhTPO and pegylated recombinant human megakaryocyte growth and development factor (PEG-rhMGDF)] rapidly entered clinical trials in 1995 and increased platelet counts in humans undergoing non-myeloablative chemotherapy but not in those undergoing stem cell transplantation. Antibodies developed against PEG-rhMGDF and development of these recombinant thrombopoietins ended. A second generation of TPO receptor agonists (romiplostim and eltrombopag) was then developed. Neither of these TPO receptor agonists demonstrated any significant untoward effects and both are now licensed in many countries for the treatment of immune thrombocytopenia. This review describes the significant experiments that have surrounded the discovery of TPO and its clinical development.

The importance of calcium in the regulation of megakaryocyte function

Platelet release by megakaryocytes is regulated by a concert of environmental and autocrine factors. We previously showed that constitutively released adenosine diphosphate by human megakaryocytes leads to platelet production. Here we show that adenosine diphosphate elicits, in human megakaryocytes, an increase in cytosolic calcium concentration, followed by a plateau, which is lowered in the absence of extracellular calcium, suggesting the involvement of Store-Operated Calcium Entry. Indeed, we demonstrate that megakaryocytes express the major candidates to mediate Store-Operated Calcium Entry, stromal interaction molecule 1, Orai1 and canonical transient receptor potential 1, which are activated upon either pharmacological or physiological depletion of the intracellular calcium pool. This mechanism is inhibited by phospholipase C or inositol-3-phosphate receptor inhibitors and by a specific calcium entry blocker. Studies on megakaryocyte behavior, on extracellular matrix proteins that support proplatelet extension, show that calcium mobilization from intracellular stores activates signaling cascades that trigger megakaryocyte adhesion and proplatelet formation, and promotes extracellular calcium entry which is primarily involved in the regulation of the contractile force responsible for megakaryocyte motility. These findings provide the first evidence that both calcium mobilization from intracellular stores and extracellular calcium entry specifically regulate human megakaryocyte functions.

Inverse Agonism of SQ 29,548 and Ramatroban on Thromboxane A2 Receptor

G protein-coupled receptors (GPCRs) show some level of basal activity even in the absence of an agonist, a phenomenon referred to as constitutive activity. Such constitutive activity in GPCRs is known to have important pathophysiological roles in human disease. The thromboxane A2 receptor (TP) is a GPCR that promotes thrombosis in response to binding of the prostanoid, thromboxane A2. TP dysfunction is widely implicated in pathophysiological conditions such as bleeding disorders, hypertension and cardiovascular disease. Recently, we reported the characterization of a few constitutively active mutants (CAMs) in TP, including a genetic variant A160T. Using these CAMs as reporters, we now test the inverse agonist properties of known antagonists of TP, SQ 29,548, Ramatroban, L-670596 and Diclofenac, in HEK293T cells. Interestingly, SQ 29,548 reduced the basal activity of both, WT-TP and the CAMs while Ramatroban was able to reduce the basal activity of only the CAMs. Diclofenac and L-670596 showed no statistically significant reduction in basal activity of WT-TP or CAMs. To investigate the role of these compounds on human platelet function, we tested their effects on human megakaryocyte based system for platelet activation. Both SQ 29,548 and Ramatroban reduced the platelet hyperactivity of the A160T genetic variant. Taken together, our results suggest that SQ 29,548 and Ramatroban are inverse agonists for TP, whereas, L-670596 and Diclofenac are neutral antagonists. Our findings have important therapeutic applications in the treatment of TP mediated pathophysiological conditions.

Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis

The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.

Developmental Differences Between Newborn and Adult Mice In Response To Romiplostim

Thrombocytopenia affects 20-35% of infants admitted to Neonatal Intensive Care Units. The incidence of thrombocytopenia is inversely proportional to gestational age, and approaches 70% among the most preterm neonates (birth weight <1,000 grams). Preterm infants also have the highest incidence of bleeding of any age group, with 25-31% developing intracranial hemorrhage. Currently, platelet (plt) transfusions are the only therapeutic option for thrombocytopenic neonates. In the last 5 years, two thrombopoietin (TPO) mimetics, romiplostim (ROM) and eltrombopag, received FDA approval for the treatment of adults with ITP. Based on the severity and duration of thrombocytopenia, 10% of thrombocytopenic neonates could benefit from TPO-mimetic therapy. Our prior in vitro studies demonstrated that human neonatal megakaryocyte (MK) progenitors are significantly more sensitive to TPO than adult progenitors (Pastos et al., Blood, 2006; Liu et al., Blood, 2011). This study was designed to compare the in vivo responses of newborn vs. adult mice to ROM. Based on prior observations, we hypothesized that newborn pups would be more sensitive to TPO-mimetics than adult mice.As a first step, healthy adult C57BL/6 mice were given a single subcutaneous (SC) injection of 0.1% BSA (control) or ROM at a dose of 10, 30, 100, or 300 ng/g body weight. Newborn mice on post-natal day 1 (P1) received a single SC injection of either 0.1% BSA or ROM at a dose of 30 or 300 ng/g. Plt count and immature plt fraction (IPF) were measured on the day of injection and every other day for 14 days. The baseline plt count in adult mice was 1,184±204 x103/µL. Adult mice treated with ROM (n=3-4 per group) exhibited a dose-dependent increase in plt count and IPF, which peaked on day 5 in those receiving lower ROM doses (10 and 30 ng/g), and on day 7 in those receiving higher ROM doses (100 and 300 ng/g). On day 7, adult mice treated with ROM 300 ng/g had a 4.2-fold increase in plt count compared to BSA controls (6,733±511 vs. 1,600±216 x103/µL, respectively; p<0.0001). Newborn mice (P1) had significantly lower baseline plt counts (624±130 x103/µL; p<0.0001) compared to adults, and similarly responded to ROM injection with a dose-dependent increase in plt count that peaked on day 5. However, plt counts on post-natal day 5 (P5) were 1,020±198 x103/µL for newborn mice treated with ROM 30 ng/g and 1,355±137 x103/µL for newborn mice treated with ROM 300 ng/g (n=17 per group), representing less than a 2-fold increase over BSA treated pups (701±119 x103/µL).To evaluate the effect of ROM on megakaryopoiesis, a subset of adult and newborn mice treated with 0.1% BSA or ROM 300 ng/g (n=3-4 per group) were euthanized on day 5 after injection. Liver, spleen, and bone marrow (BM) MKs were immunohistochemically stained for von Willebrand factor and quantified as described (Hu Z et al., Neonatology, 2010). Overall, ROM-treated adult mice had significantly increased numbers of MKs compared to controls in BM (2.3-fold increase; p=0.0002) and spleen (3.9-fold increase; p=0.006). ROM-treated newborn mice exhibited non-significant increases in MK numbers in BM (2.2-fold increase; p=0.19), spleen (1.6-fold increase; p=0.35), and liver (1.4-fold increase; p=0.31).Because newborn C57BL/6 mice transition from fetal liver to adult BM hematopoiesis during the first 10 to 14 days of life and the BM is not well formed until P10, we injected newborn mice at P5 (instead of P1) and evaluated the response to ROM. Similar to the younger group, P5 mice treated with ROM 300 ng/g reached peak platelet counts at P11, but the plt count was only 1.4-fold higher than BSA control animals (1,340±440 vs. 927±151 x103/µL, respectively; p=0.19).In conclusion, this study indicated that newborn mice are less responsive to ROM than adult mice. This was a surprising finding, given that human neonatal MK progenitors have been consistently shown to be more sensitive to TPO than adult MK progenitors. The reasons underlying the modest in vivo response of neonates are unclear, but might be related to the transition in hematopoietic sites that occurs during this period in murine development (corresponding to the second trimester of human gestation), high baseline thrombopoietic demands associated with rapid growth, potential pharmacokinetic factors, or developmental differences in the splenic or BM microenvironments of newborn and adult mice. 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.

Development Of 3D Models For Studying Megakaryopoiesis In Vitro

IntroductionSilk fibroin, derived from Bombyx mori silkworm cocoons, is used extensively in biomaterials and regenerative medicine. The useful characteristics of this protein include self-assembly, robust mechanical properties, biocompatibility and biodegradability. Moreover, silk can be enhanced through a variety of chemical modifications that affect cell attachment, growth and differentiation. Thrombocytopenia occurs when a patient suffers from an abnormally low platelet count in the peripheral blood; usually a result of disease, trauma, or cancer treatment. To treat these patients, it is estimated that two million platelet transfusions are performed in the U.S. each year. This high demand for platelets has created a clinical demand for studying the causes of thrombocytopenia and alternative routes for treatment. Platelets are anuclear cells that are released into the bloodstream in the bone marrow by megakaryocytes via the extension of long filaments called proplatelets. It is hypothesized that platelet production from megakaryocytes is regulated by environmental factors at the site of bone marrow vascular niche. Studies of megakaryopoiesis are typically performed on extracellular matrix protein-coated culture plates and transwell membranes. While these initial studies have provided invaluable insight into the process of megakaryopoiesis, the goal of the present project was to create a bone marrow model that mimics the vascular niche for functional in vitro platelet production. We hypothesized that a silk-based in vitro tissue model would allow the effects of substrate surface properties and endothelial co-culture on megakaryopoiesis to be studied in a holistic manner, thereby enabling further elucidation of the mechanisms involved in the process of platelet production. ResultsIn order to more closely mimic the bone marrow vascular niche structure, a porous silk sponge was assembled around the silk vessel-like tubes. Megakarycytes seeded in the porous silk sponge migrated toward the silk tube and released platelets into the tube lumen. The perfusion bioreactor moved the platelets into the platelet collecting bags. After perfusion the platelets were collected and analyzed by flow cytometry. The bioreactor platelets exhibited similar morphology, CD41 positive staining, and activation compared to peripheral blood platelet controls. Megakaryocyte attachment and proplatelet formation through the silk vascular wall were improved by altering the silk properties. Silk functionalized by entrapping extracellular matrix proteins within the tube membrane resulted in increased megakaryocyte attachment and proplatelet compared to unfunctionalized silk tube controls. Silk surface roughness improved megakaryocyte attachment compared to the control but did not affect proplatelets. Decreasing the silk stiffness improved proplatelets, but did not significantly affect megakaryocyte attachment. Co-culture with endothelial cells improved megakaryocyte attachment while maintaining a high level of proplatelet formation. Additionally, megakaryocyte and endothelial cell co-culture on the silk vessel model resulted in an icreased platelet production compared to megakaryocytes cultured alone. ConclusionsThe goal of this project was to develop an in vitro model of megakaryopoiesis using a tissue engineering approach. Using human megakaryocytes and endothelial cells, we demonstrate the following advanced features of the silk-based model: (1) immobilization of extracellular matrix components within the membrane, (2) tunable surface topography, (3) tunable mechanical properties, (4) physiologically relevant thickness for appropriate proplatelet extension, and (5) controlled localization of a vascular endothelium. Thus, by functionalizing silk, we can control megakaryocyte function on silk. The broader impact of this work offers a versatile new tool for studying megakaryocyte development and platelet production in vitro. Disclosures:No relevant conflicts of interest to declare.

Effects Of TPO, Romiplostim and Eltrombopag On Neonatal Vs. Adult Human Megakaryopoiesis

IntroductionThrombocytopenia affects 20-30% of infants admitted to Neonatal Intensive Care Units. In 20-25% of affected neonates, the platelet count drops to <50x109/L, and 9% of those infants experience clinically significant bleeding. Currently, platelet transfusions are the only therapeutic option for thrombocytopenic neonates. Two novel thrombopoietin (TPO) mimetics, romiplostim (ROM) and eltrombopag (ELT), are in clinical use for the treatment of adults with ITP. Based on the severity and duration of thrombocytopenia, 10% of thrombocytopenic neonates could benefit from this therapy. We have previously described significant cellular and molecular differences between neonatal and adult megakaryocytes (MKs) in their response to TPO (Pastos et al., Blood 2006; Liu et al., Blood 2011). Thus, developmental stage-specific studies are needed prior to considering the use of these agents in neonates. This study was designed to compare the responses of human neonatal vs. adult MK progenitors to TPO, ROM, and ELT in vitro. MethodsHematopoietic progenitor (CD34+) cells isolated from cord blood (CB) and adult mobilized peripheral blood (PB) were cultured in a serum-free culture system in the presence of escalating concentrations of rTPO (1-100 ng/ml), ROM (1-200 ng/ml), or ELT (1-30 µM). In each dose-response experiment, cells were also cultured with 50 ng/mL of rTPO as an internal control. After 14 days, cells were counted and their differentiation (CD41 expression), maturation (CD42b expression) and ploidy were evaluated by three-color flow cytometry. To adjust for inter-individual differences, the responses to escalating concentrations of ROM or ELT were expressed as percentages of the number of MKs cultured with 50 ng/mL of TPO (“percent of TPO50”). ResultsConsistent with our prior studies, CB MK progenitors proliferated at a higher rate than adult PB progenitors in response to TPO, and generated approximately 10 times more MKs (CD41+ cells) per CD34+ cell than adult PB (CB n=3, PB n=4). To compare the sensitivity of CB and PB MK progenitors to ROM and ELT, percent of TPO50 vs. ROM or ELT concentration curves were generated. CB progenitors (n=3) generated the highest number of MKs at 200 ng/mL of ROM, which was nearly equivalent to TPO50. PB progenitors (n=2), in contrast, generated the maximal MK number at 30 ng/mL of ROM, and this represented approximately 80% of TPO50. At all concentrations of TPO and ROM, CB MKs exhibited lower ploidy levels than PB MKs (4-10% vs. 16-34% MKs ≥8n, respectively), but higher percentages of CD42+ MKs (85-95% vs. 63-80% in CB and PB, respectively). Next, we generated percent of TPO50 vs. ELT concentration curves. CB progenitors (n=5) reached the highest MK number at 6 µM ELT, and this represented approximately 40% of TPO50. For PB MKs (n=4), the maximal number of MKs was generated at 10 µM ELT, and this represented only 10% of TPO50. Nevertheless, these MKs had ploidy and CD42 expression levels similar to those cultured in TPO50. ELT concentrations higher than 10 µM did not support the survival of MK progenitors. To evaluate whether this toxicity was TPO receptor-dependent or not, we then cultured CD34+ cells with escalating ELT concentrations together with a physiological concentration of TPO (CB n=3, PB n=2). These studies revealed a strong synergism between TPO and low concentrations of ELT, particularly in CB cultures. However, PB cells proliferated less and CB cells died in the presence of higher ELT concentrations (30 µM) despite the addition of TPO, suggesting that the dose-dependent ELT toxicity is TPO receptor-independent. ConclusionsOverall, we confirmed our previous findings that CB progenitors are hyperproliferative compared to adult progenitors, and generate small, low-ploidy, but mature MKs. Dose-response curves were similar for TPO and ROM. ELT generated less MKs than TPO or ROM, although these MKs were fully mature. CB MKs were more sensitive than PB MKs to the thrombopoietic as well as to the toxic effects of ELT. The dose-dependent toxicity of ELT in vitro is TPO-receptor independent. The mechanisms underlying this effect are unknown, but might be related to the structural similarities between ELT and metal chelators, potentially inducing intracellular iron depletion as has been shown in leukemic cells (Roth et al., Blood 2012). Disclosures:No relevant conflicts of interest to declare.

A Dominant-Negative GFI1B Mutation in Gray Platelet Syndrome

Gray platelet syndrome (GPS) is a hereditary, usually autosomal recessive bleeding disorder caused by defective production of α-granules in platelets. GPS patients show reduced numbers of platelets that are larger and have a typical gray appearance under light microscopy, caused by the lack of α-granules. We describe a large family with an autosomal dominant type of GPS characterized by mild to severe bleeding complications. In addition to large gray platelets, other GPS-associated phenomena like myelofibrosis, thrombocytopenia, and low platelet factor 4 expression were observed in affected individuals. Histopathological examination of a BM biopsy from a patient showed a cellular marrow with increased numbers of megakaryocytes that were pleomorphic in size and shape. Megakaryocytes clustered along BM sinuses and showed dysmorphic stretched features.To determine the disease causing mutation we performed linkage analysis and identified a candidate locus on chromosome 9q34 with a LOD score of 3.9. We considered GFI1B (Growth Factor Independence 1B), located within this region, an excellent candidate gene because of its function as a transcriptional repressor in megakaryocyte development. Sequence analysis identified a nonsense mutation in GFI1B exon 6 (c.859C>T, p.Gln287*) that completely co-segregated with the GPS disease in this family. The mutated transcript predicts a 44 amino acid C-terminally truncated protein, GFI1BTr. The truncation is located within zinc finger 5 of GFI1B, deleting all of its four amino acids that directly interact with DNA. Luciferase gene reporter assays showed that GFI1BTr was unable to repress gene expression. Importantly, GFI1BTr inhibited gene repression mediated by wild type GFI1B, indicating that the mutant interferes with wild type GFI1B in a dominant-negative manner. To validate that GFI1BTr adversely affects normal GFI1B, we expressed the mutant in mouse bone marrow cells followed by induction of megakaryocytic differentiation. Compared to control cells, GFI1BTr-positive megakaryocytes showed dysplastic features including hypolobulation of the nuclei, irregular contours and multiple separate nuclei, that were very similar to those observed in patient cells. This indicates that GFI1BTr causes megakaryocytic abnormalities and that it functions in a dominant-negative manner.GFI1B silencing inhibits the development of human megakaryocyte colonies in vitro. We observed that megakaryocyte colony forming cells were significantly more frequent in patient bone marrow compared to controls. In addition, patient-derived megakaryocyte colonies were significantly larger compared to controls. Immunophenotypic analyses of peripheral blood showed no differences in myeloid and erythroid lineages and the platelet markers GP3B, ITGA2B and ITGB3 among affected an non-affected individuals. However, within the ITGA2B/CD41-positive platelet population, 5 of 6 affected members showed a marked decrease in the platelet surface membrane glycoprotein 1b-α (GP1BA/CD42b), compared to unaffected members. In addition, a strong expression of CD34, which is usually confined to immature hematopoietic progenitors, was detected on platelets from all studied affected individuals. Immunostaining of a BM biopsy from a patient showed the presence of ITGB3/CD61 positive megakaryocytes that intensely expressed CD34. Electron microscopy analysis showed megakaryocytes with few, small, irregularly shaped and centrally located α-granules characterized by an extensive peripheral cytoplasm with irregular proplatelets, largely devoid of cell organelles. To test whether these abnormalities were cell intrinsic, we stimulated CD34+ cells from two patients to differentiate along the megakaryocytic lineage in vitro. Megakaryocytic cells showed dysplastic features reminiscent of those observed in the bone marrow aspirates. In addition, increased CD34 and decreased GP1BA/CD42b expression were observed on megakaryocytes, indicating that GFI1BTr-induced abnormalities are intrinsic to the cell. In summary, we have identified GFI1B as a causative gene in autosomal dominant GPS. GFI1BTr acts in a dominant-negative manner over wild type GFI1B and affects the development of megakaryocytes and platelets, demonstrating a pivotal role of GFI1B in governing normal megakaryopoiesis and platelet production. Disclosures:No relevant conflicts of interest to declare.