c-Mpl Expression Research Articles

Sox17 as a candidate regulator of myeloid restricted differentiation potential.

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and β-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.

Properties of c-Mpl expression in thrombopoietin-derived hepatic hematopoietic progenitors of xenopus laevis

P1116 PROPERTIES OF C-MPL EXPRESSION IN THROMBOPOIETINDERIVED HEPATIC HEMATOPOIETIC PROGENITORS OF XENOPUS LAEVIS Yoko Mochizuki, Yuta Tanizaki, Takato Otani, Kei Sato, Megumi Ichisugi, Ayaka Murase, Shunji Sakai, and Takashi Kato Graduate School of Advanced Science and Engineering, Waseda University, Tokyo, Japan; Department of Biology, School of Education, Waseda University, Tokyo, Tokyo, Japan

Serum Thrombopoietin and cMpl Expression in Thrombocytopenia of Different Etiologies

The relationship between thrombopoietin (TPO) and its receptor cMpl in thrombocytopenic conditions has not been entirely clarified. To elucidate this interplay may expand the spectrum of indications of TPO mimetics. In this study we have explored the relationship between TPO and cMpl in platelets and megakaryocytes of 43 patients with thrombocytopenia due to idiopathic thrombocytopenic purpura (ITP), bone marrow hypoplasia, myelodysplastic syndromes (MDS), and familial thrombocytopenia. Data were compared to cMpl and TPO in patients with a normal platelet count and in patients with thrombocytosis due to essential thrombocythemia (ET). All but familial patients showed higher TPO compared to controls. All thrombocytopenic states were invariably associated with increased expression of platelet cMPL compared to healthy controls. ET patients showed normal TPO and a trend toward a reduced cMpl expression. Immunofluorescence of bone marrow sections from patients with ITP and MDS failed to show a peculiar pattern compared to controls. Multiple mechanisms regulate TPO and cMpl in thrombocytopenic conditions.

Thrombopoietin from beginning to end

In the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench-to-bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan-haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c-Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non-redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c-Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research.

Posttranslational Modification Of The Thrombopoietin Receptor Regulates Cytokine Signal Transduction and Hematopoietic Stem Cell Engraftment

BackgroundThrombopoietin (TPO) is a key cytokine regulating hematopoietic stem cell (HSC) quiescence and self-renewal, and has been shown to augment ex vivo expansion and maintenance of HSCs in culture. Impaired TPO signaling is associated with bone marrow failure and thrombocytopenia. TPO mediates intracellular signal transduction by binding its cell surface receptor, cMpl, and transgenic mice deficient in cMpl expression have an age-progressive reduction in HSC content. An improved understanding of the mechanisms regulating HSC proliferation, self-renewal, and quiescence is essential to optimize transplantation, expansion, and other therapeutic applications of HSCs.Posttranslational modification of proteins in the Golgi results in conjugation of complex-type N-glycans on glycoproteins destined for the cell surface. A complete deficiency of glycosylation results in failure of transport of proteins to the plasma membrane, while glycosylation with complex branched carbohydrates regulates a wide variety of functions at the cell surface including receptor clustering, cross-linking and trafficking. N-glycosylation of tyrosine kinase cytokine receptors has been shown to enhance their signaling potential by sparing receptor internalization via endocytosis, thus promoting receptor retention at the cell surface (Partridge EA et al, Science 2004). cMpl is known to undergo N-linked glycosylation, with defects in posttranslational processing associated with polycythemia vera. However, little is known about the functional significance of glycosylation of cMpl on TPO signaling and HSC function.Here we show that glycosylation of cMpl regulates TPO signal transduction in an endocytosis-dependent manner, resulting in enhanced short-term engraftment in a mouse model of in utero hematopoietic cell transplantation (IUHCT). Methods293T cells stably overexpressing the TPO receptor cMpl were treated with either the α-mannosidase II inhibitor swainsonine (1 μg/ml) to inhibit complex N-glycan branching or the hexosamine biosynthetic pathway substrate UDP-GlcNAc (12.5 – 50 mM) to increase N-glycan production. Activation of signaling proteins was measured by immunoblotting and immunofluorescence studies. Clathrin- and caveolin-dependent endocytosis was inhibited by potassium depletion and nystatin treatment, respectively. The effect of posttranslational cMpl modification on donor cell engraftment was assessed in a murine model of IUHCT. Fetal liver from gestational day 14 Balb/c recipient fetuses were assessed for donor cell chimerism 48 hours after transplantation with 10e6 B6-GFP bone marrow cells with or without UDP-GlcNAc treatment. ResultsInhibition of N-linked glycosylation by swainsonine treatment resulted in reduced downstream signal transduction following TPO stimulation, with significantly decreased levels of phosphorylation of MAPK (Figure 1A). Pharmacologic disruption of endocytosis was found to ablate the effects of swainsonine treatment, with equivalent and robust activation of MAPK in swainsonine-treated and untreated cells. Upregulation of N-linked glycosylation by supplementation of the glycosylation pathway with UDP-GlcNAc treatment resulted in a dose-dependent increase in signaling following TPO stimulation, with increased levels of phosphorylation of MAPK (Figure 1B). Finally, UDP-GlcNAc treatment of donor cells resulted in significantly higher engraftment levels at 48 hours post IUHCT compared to controls (20.18 +/- 2.77 versus 30.27 +/- 1.985, p=0.0417). [Display omitted] ConclusionsThese results support a role for cMpl glycosylation in modulating TPO signal transduction in an endocytosis-dependent manner, and suggest that enhanced cMpl glycosylation can optimize HSC function resulting in increased levels of engraftment following IUHCT. Ongoing studies of receptor trafficking, including internalization by clathrin-dependent and –independent endocytosis, are needed to further elucidate the functional significance of complex N-glycans on cMpl fate. These studies will ultimately improve our understanding of how TPO signaling may be manipulated to control HSC self-renewal and quiescence to optimize in vitro expansion and HSC transplantation. Disclosures:No relevant conflicts of interest to declare.

Increased Angiogenic Property Of Human Peripheral Blood Monocytes By ex Vivo Culture With c-Mpl Agonists In Hindlimb Ischemia Mouse Model

Human peripheral blood mononuclear cells (PB-MNCs) include some populations which have angiogenic properties. Pro-angiogenic monocytes from PB-MNCs are considered as one of candidates for angiogenic therapy in regenerative medicine. Indeed, in a recent German clinical post-infarction remodeling study (TOPCARE-AMI) for ischemic heart disease, the ex vivo culture of PB-MNCs was employed. However, in this trial, there were different therapeutic efficacies in each case, possibly due to the different expansion efficacy of the ex vivo culture of PB-MNCs using autologous serum.In order to resolve this issue, we developed a new serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Floating spheres obtained by this serum-free culture system were mainly composed of CD11b+ monocytes. Interestingly, the mRNA expression of c-Mpl (thrombopoietin receptor) was markedly elevated compared with PB-MNCs, suggesting c-Mpl agonist could increase angiogenic property of cultured CD11b+ monocytes. Therefore, we assessed the impact of c-Mpl agonists on PB-MNC cultures in our serum-free method composed of X-VIVO15 medium with VEGF and bFGF.Both recombinant human thrombopoietin (rHuTPO) and romiplostim, a clinical grade second-generation TPO-receptor agonist, successfully increased sphere formations regarding both the number and size. The expressions of angiogenic factors, IL-8, CXCR4, and vasohibin-2, mRNA of CD11b+ monocytes cultured with c-Mpl agonists were up-regulated, indicating that cultivated CD11b+ monocytes have a proangiogenic potential.Finally, we investigated the proangiogenic potential of PB-MNCs derived CD11b+ monocytes in a hindlimb ischemia model utilizing BALB/c nude mice. Mice were randomly assigned to 7 groups: control mouse group (PBS-injected), freshly isolated CD11b+ monocyte-injected mouse group, cultivated CD11b+ monocyte with 2ng/ml and 20ng/ml rHuTPO -injected mouse groups, cultivated CD11b+ monocyte with 100ng/ml and 1000ng/ml romiplostim -injected mouse groups, cultivated CD11b+ monocyte without rHuTPO and romiplostim -injected mouse group. The intramuscular injection of CD11b+ monocytes cultivated with 20 ng/ml rHuTPO into the ischemic limb completely rescued the limbs from auto-amputations or foot necrosis, while only one (10.0%) of the control mice could be rescued. In addition, the intramuscular injection of both freshly isolated CD11b+ monocytes and CD11b+ monocytes cultivated without rHuTPO and romiplostim had a weak rescue effect on the ischemic limbs (8 and 7 of 10 mice had auto-amputations or foot necrosis, respectively). The salvage rate from necrosis in cultivated CD11b+ monocyte with romiplostim-injected mouse group is also superior to that in cultivated CD11b+ monocyte without rHuTPO-injected mouse group. Analysis of blood perfusion by a laser Doppler perfusion imaging system showed a significantly higher recovery in mice receiving the CD11b+ monocytes cultivated with 2 ng/ml or 20 ng/ml rHuTPO or 100ng/ml romiplostim 1 week after surgery. The functional capillary density and surface area visualized by perfusion with BS-I lectin also significantly increased in the rHuTPO- or romiplostim-treated group.In conclusion, an ex vivo addition of c-Mpl agonists augmented the pro-angiogenic activity of peripheral CD11b+ monocytes, and this method would be promising for human cell therapy to induce vascular regeneration by activating the angiogenic property in human peripheral blood-derived monocytes. Disclosures:Mizukami:The New Energy and Industrial Technology Development Organization of Japan: Research Funding.

Ott1(Rbm15) Regulates Thpo Response In HSCs Through Epigenetic Modifications Directing Alternative Splicing Of C-Mpl

Thrombopoietin (Thpo), through its receptor c-Mpl, is essential for Hematopoietic Stem Cell (HSC) function and has a dose-dependent effect in which low concentrations promote quiescence and self-renewal in contrast to high Thpo concentrations which promote proliferation. Thpo production is largely stable in vivo, therefore it is unclear how this dual response is evoked physiologically. HSCs deleted for c-Mpl are unable to tolerate proliferative stress. Ott1(Rbm15), the 5' fusion partner in t(1;22) acute megakaryocytic leukemia, is also essential for maintaining HSC quiescence during proliferative stress, however the mechanism has not been elucidated. Total c-Mpl expression in Ott1-deleted HSCs does not significantly differ from wild type, however, the existence of a cross-species, conserved isoform, Mpl-TR, with dominant negative activity, suggests a potential mechanism for affecting c-Mpl signaling via alternative splicing. Ott1 is a spliceosome component, is implicated in RNA processing and possesses RNA Recognition Motifs, yet has not been linked with any known physiologic targets. Analysis of c-Mpl isoforms in HSC-containing Lin-Sca1+c-Kit+ fractions and fetal liver megakaryocytes showed a marked increase in the ratio of Mpl-TR transcript. Ott1-deleted HSC populations displayed reduced Stat5 phosphorylation in response to Thpo stimulation consistent with decreased Mpl signaling. Exogenous expression of Mpl-TR in wild type bone marrow dramatically reduced short and long term engraftment into irradiated recipients, confirming in vivo activity of Mpl-TR in HSCs. To determine whether Ott1 complexes with Mpl RNA, RNA-immunoprecipitation was performed using an HA-tagged Ott1 and revealed complex formation with Mpl RNA. Alternative splicing is frequently regulated through a co-transcriptional mechanism utilizing local epigenetic modifications including histone acetylation and H3K4me3 marks. Ott1 was previously shown to bind class I Histone deacetylases (Hdacs) and the histone H3K4 methyl-transferase (HMT), Setd1b. To establish whether Ott1 interacts with the c-Mpl gene, Chromatin-immunoprecipitation (ChIP) using HA-tagged Ott1 was performed and found binding within regions flanking the alternatively spliced exons. ChIP using anti-pan-acetyl-H4 in Ott1 knockout Lin- bone marrow showed increased histone acetylation in the region shown to bind Ott1 compared to wild type. Conversely, ChIP using anti-H3K4me3 in the Ott1 knockout showed decreased H3K4me3 at the site of Ott1 binding consistent with loss of Ott1-associated Hdac and HMT activity. To test the functional consequences on splicing, treatment of wild type cells with either a class I Hdac inhibitor or a HMT inhibitor was able to significantly increase the ratio of Mpl-TR isoform.In summary, Ott1 regulates the production of the alternatively spliced c-Mpl isoform, Mpl-TR, and consequently Thpo response in HSCs. Mpl-TR expression impairs physiologic HSC function for long and short term engraftment. Ott1 complexes with c-Mpl RNA and chromatin adjacent to the exons alternatively spliced in the Mpl-TR isoform and regulates histone acetylation and methylation marks associated with splice decision. Therefore, Ott1-mediated alternative splicing of Mpl may provide a novel mechanism via chromatin modification for modulating HSC maintenance and proliferation in response to Thpo. Furthermore, the ability to control Mpl alternative splicing through epigenetic inhibitors opens unique possibilities for pharmacologically manipulating HSC function in vitro or in vivo. Disclosures:No relevant conflicts of interest to declare.

The TPO/c-MPL Pathway in the Bone Marrow may Protect Leukemia Cells from Chemotherapy in AML Patients

Accumulating evidence indicates that the interaction of human LSCs (leukemic stem cells) with the hematopoietic microenvironment, mediated by the thrombopoietin (TPO)/c-MPL pathway, may be an underlying mechanism for resistance to cell cycle-dependent cytotoxic chemotherapy. However, the role of TPO/c-MPL signaling in AML (acute myelogenous leukemia) chemotherapy resistance hasn't been fully understood. The c-MPL and TPO levels in different AML samples were measured by flow cytometry and ELISA. We also assessed the TPO levels in the osteoblasts derived from bone mesenchymal stem cells (BMSCs). The survival rate of an AML cell line that had been co-cultured with different BMSC-derived osteoblasts was measured to determine the IC50 of an AML chemotherapy drug daunorubicin (DNR). The levels of TPO/c-MPL in the initial and relapse AML patients were significantly higher than that in the control (P < 0.05). The osteoblasts derived from AML patients' BMSCs secreted more TPO than the osteoblasts derived from normal control BMSCs (P < 0.05). A strong positive correlation between the TPO level and c-MPL expression was found in the bone marrow mononuclear cells of the relapse AML patients. More importantly, the IC50 of DNR in the HEL + AML-derived osteoblasts was the highest among all co-culture systems. High level of TPO/c-MPL signaling may protect LSCs from chemotherapy in AML. The effects of inhibition of the TPO/c-MPL pathway on enhancing the chemotherapy sensitivity of AML cells, and on their downstream effector molecules that direct the interactions between patient-derived blasts and leukemia repopulating cells need to be further studied.

Novel Anti-c-Mpl Monoclonal Antibodies Identified Multiple Differentially Glycosylated Human c-Mpl Proteins in Megakaryocytic Cells But Not in Human Solid Tumors

Thrombopoietin and its cognate receptor, c-Mpl, are the primary molecular regulators of megakaryocytopoiesis and platelet production. To date the pattern of c-Mpl expression in human solid tumors and the distribution and biochemical properties of c-Mpl proteins in hematopoietic tissues are largely unknown. We have recently developed highly specific mouse monoclonal antibodies (MAb) against human c-Mpl. In this study we used these antibodies to demonstrate the presence of full-length and truncated human c-Mpl proteins in various megakaryocytic cell types, and their absence in over 100 solid tumor cell lines and in the 12 most common primary human tumor types. Quantitative assays showed a cell context-dependent distribution of full-length and truncated c-Mpl proteins. All forms of human c-Mpl protein were found to be modified with extensive N-linked glycosylation but different degrees of sialylation and O-linked glycosylation. Of note, different variants of full-length c-Mpl protein exhibiting differential glycosylation were expressed in erythromegakaryocytic leukemic cell lines and in platelets from healthy human donors. This work provides a comprehensive analysis of human c-Mpl mRNA and protein expression on normal and malignant hematopoietic and non-hematopoietic cells and demonstrates the multiple applications of several novel anti-c-Mpl antibodies.

Pre-Adipocytes Differentiate Into Megakaryocytes (MK) by a Paracrine Thrombopoietin (TPO) Loop

Abstract 4742The regulation of megakaryopoiesis and thrombopoiesis are incompletely understood. Better understanding of the underlying mechanisms would be of biological import, but may also lead to novel approaches for generating of platelets for clinical application. One cell line that undergoes terminal differentiation into megakaryocytes is pre-adipocytes. These cells represent a potential candidate cell for ex vivo generation of megakaryocytes. Here we demonstrate that pre-adipocytes differentiate into MKs and platelets by using an endogenous paracrine loop involving TPO, the primary cytokine involved in megakaryopoiesis, and its receptor c-mpl. We previously reported that pre-adipocytes differentiate into MKs and form platelets when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium. Neither media include TPO. Based on these observations, the present study tested the hypothesis that pre-adipocytes might differentiate into MKs and platelets by endogenous TPO and c-mpl expression of sufficient magnitude to drive megakaryopoiesis. We used primary mouse pre-adipocytes (CD45-, CD117-, Sca1+, CD29+, CD73+, CD90+, CD105+, CD106+) from subcutaneous adipose tissues and also the mouse pre-adipocyte line 3T3-L1 cells. The TPO levels, as assessed by ELISA, in the supernatant from 106 pre-adipocytes cultured in 2 ml MKLI medium without exogenous TPO (TPO-) were unmeasurable level on Day 0, 29±14 pg/ml on Day 7 and 8±2 pg/ml on Day 12. Similar results were obtained in the supernatant from 3T3-L1 during MK differentiation. We did not observe measureable TPO in supernatants from pre-adipocytes and 3T3-L1 cells cultured in maintenance medium on Days 0, 7 and 12. We then compared MK differentiation from pre-adipocytes in MKLI media in the absence (TPO-) or addition (final concentration, 50 ng/ml; TPO+) of TPO. The frequency of CD41-postive pre-adipocytes in culture after 6 days was 58±11% for TPO+ and 70±7% for TPO- (p>0.05), consistent with endogenous TPO being sufficient under these circumstances to stimulate MK differentiation of pre-adipocytes. DNA ploidy and c-mpl expression assessed by immunohistochemistry were also similar with or without added exogenous TPO. We next examined the number of CD41-expressing large-sized (>15 μm) cells beginning with 1.2 × 104 preadipocytes in the absence or presence of the c-mpl inhibitor rat anti-mouse c-mpl inhibitor AMM2 (final concentration, 10 μg/ml). In the absence of AMM2, 10-fold more CD41-positive, large cells were seen (6.8±1.7×103) than in its presence (0.6±0.2×103, p<0.05). These observations support that pre-adipocytes differentiate into MKs using endogenous TPO stimulation via c-mpl. We next examined the ability to release platelets from these treated pre-adipocytes in each experimental condition (pre-adipocytes and 3T3-L1 cells, each ± exogenous TPO). Infusion of 2 × 105 carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled MKs into irradiated thrombocytopenic mice (7 days post-2.0Gy exposure) led to a time-dependent appearance of CFSE+/CD41+ platelet-sized particles with a peak at 4 hours after the infusion reaching ∼2.5% of the total circulating platelet population (∼30 platelets/infused MK) under all tested experimental conditions. For platelet function, blood samples from each of these mice were perfused over a collagen-coated chip for 10 minutes, and the incorporation of CFSE+ particles into thrombi on the chip was determined (Total Thrombus-formation Analyzing System). A similar degree of platelet incorporation was observed under all experimental conditions and each was with an efficiency similar to that seen when CFSE+ control platelets were infused. These findings demonstrate that pre-adipocytes differentiate into MKs and subsequent platelets by an endogenous TPO release and a paracrine loop involving c-mpl. We propose that such pre-adipocytes could be used as a model of a continuously replicating cell line that upon switching media simultaneous expresses TPO and its receptor c-mpl to establish a paracrine loop leading to terminal differentiation into functional MKs. The basis of why this media switch induces this change needs to be clarified to further develop this pre-adipocyte strategy as a donor-independent source for platelet transfusion as well as for studying mechanism underlying megakaryopoiesis and thrombopoiesis. Disclosures:Matsubara:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.

Rescue of the HSC Maintenance Defects in Ku70-Deficient Mice by Overexpression of Bcl2 Reveals a Novel Role of Bcl2 in HSC

AbstractAbstract 1235Ku70 is a key component of the non-homologous end joining (NHEJ) pathway; Ku70-deficient mice are hypersensitive to radiation and show a leaky SCID phenotype. We find that HSC from Ku70-deficient mice are severely impaired in maintenance, defective in self-renewal, competitive repopulation and BM hematopoietic niche occupancy. HSCs from Ku70-deficient mice are not prone to spontaneous apoptosis, however, they display more active proliferation, and fewer cells in a quiescent state than HSC obtained from WT mice. Further, gene expression profiling showed that multiple HSC quiescence- related genes such as c-MPL and p57 were significantly downregulated in Ku70-deficient HSCs. These data suggested that loss of quiescence results in the dramatic defect in the maintenance of Ku70-deficient HSC.Bcl2 plays important roles in both anti-apoptosis and anti-proliferation, while the anti-apoptosis function of Bcl2 has been extensively studied and well established in various settings, including HSCs, the anti-proliferation function of Bcl2 in HSC has yet to be investigated. To exam whether overexpression of Bcl2 can rescue the HSC defect in Ku70-deficient mice, H2K-Bcl2 transgenic/Ku70-deficient double mutant mice were generated. Though overexpression of Bcl2 does not rescue the SCID phenotype in Ku70-deficient mice, overexpression of Bcl2 in Ku70-deficient HSCs almost completely rescued the impaired HSC quiescence, repopulation and BM hematopoietic niche occupancy capacities. At the transcriptional level, overexpression of Bcl2 restored the expression of c-MPL and p57 comparable to WT levels. However, additional deletion of Bax, a pro-apoptosis protein in the Bcl2 family, in Ku70-deficient mice could not rescue the HSC repopulation defect, suggesting that apoptosis is not the main mediator for the impaired HSC maintenance in Ku70-deficient mice.Together, our data indicate that the HSC maintenance defect of Ku70-deficient mice is due to the loss of HSC quiescence populations, whereas overexpression of Bcl2 rescues the HSC defect in Ku70-deficient mice by restoration of quiescence. Our study uncovers a novel role of Bcl2 in hematopoietic stem cell quiescence regulation. Disclosures:No relevant conflicts of interest to declare.

Eltrombopag inhibition of acute myeloid leukemia cell survival does not depend on c-Mpl expression

Eltrombopag inhibition of acute myeloid leukemia cell survival does not depend on c-Mpl expression

The Microtubule Plus-End Tracking Protein CLASP2 Is Required for Hematopoiesis and Hematopoietic Stem Cell Maintenance

Mammalian CLASPs are microtubule plus-end tracking proteins whose essential function as regulators ofmicrotubule behavior has been studied mainly incultured cells. We show here that absence of murine CLASP2 invivo results in thrombocytopenia, progressive anemia, and pancytopenia, due to defects in megakaryopoiesis, in erythropoiesis, and in the maintenance of hematopoietic stem cell activity. Furthermore, microtubule stability and organization are affected upon attachment of Clasp2 knockout hematopoietic stem-cell-enriched populations, and these cells do not home efficiently toward their bone marrow niche. Strikingly, CLASP2-deficient hematopoietic stem cells contain severely reduced mRNA levels of c-Mpl, which encodes the thrombopoietin receptor, an essential factor for megakaryopoiesis and hematopoietic stem cell maintenance. Our data suggest that thrombopoietin signaling is impaired in Clasp2 knockout mice. We propose that the CLASP2-mediated stabilization ofmicrotubules is required for proper attachment, homing, and maintenance of hematopoietic stem cells and that this is necessary to sustain c-Mpl transcription.

C-Mpl Is Not Expressed or Functional At Detectable Levels on Primary Chronic Lymphocytic Leukemia (CLL) Tumor Samples

Abstract 2849The development of thrombocytopenia is associated with progression of CLL to a more advanced stage and is commonly used to classify CLL into the higher disease risk categories. It has been estimated that 5% of CLL patients will present with associated immune thrombocytopenia purpura (ITP) and immune mediated destruction can exacerbate thrombocytopenia related to bone marrow infiltration. Thromopoietin (TPO) mimetics stimulate platelet production though activation of the thrombopoietin receptor (c-Mpl) on megakaryocytes and are currently indicated for chronic ITP. In principle, this approach could be considered for treatment of ITP in CLL patients. In order to rule out the possibility that c-Mpl is functionally expressed in CLL tumor cells; we analyzed expression and TPO-dependent function in primary patient samples.We have developed assays for the analysis of c-Mpl expression on the surface of B-cells in CLL patient samples and to assess functional response to ex-vivo stimulation of theses primary samples with TPO. Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. This cohort included treatment naïve CLL samples (n=57) and CLL samples collected from subjects undergoing active treatment (n=30). CLL cells were analyzed by flow cytometry and identified based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a novel c-Mpl-specific monoclonal antibody that was shown to be specific for c-Mpl by flow cytometry. Residual platelets associated with individual PBMC aliquot served as an internal control for c-Mpl expression. To investigate c-Mpl function, CLL cells were stimulated with rhTPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured to assess a functional response to ex-vivo stimulation of TPO. In addition c-Mpl mRNA expression was surveyed in CLL by using mRNA microarray analyses to correlate c-Mpl mRNA expression with protein and functional expression of c-Mpl.Robust c-Mpl protein expression was observed in platelets from normal and CLL patients (fold over control -normal: 31.90 ± 6.39, CLL: 26.76 ± 6.57; mean ± 95%CI), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (fold over control −1.06 ± 0.021; mean ± 95%CI). No induction of STAT5 phosphorylation was detected in B-cell CLL cells in any of the samples stimulated with TPO (fold over control - normal: 0.90 ± 0.02, CLL: 1.04 ± 0.034; mean ± 95%CI). Microarray analysis of the CLL samples demonstrated c-Mpl mRNA expression levels equivalent to background across all CLL samples analyzed (intensity score −10.3 ± 3.0; mean ± 95%CI). In conclusion, we demonstrated a lack of cell surface c-Mpl protein expression in CLL cells. The additional data suggesting the lack of evidence for significant expression of c-Mpl mRNA expression supports the hypothesis that CLL cells do not express c-Mpl and unlikely to be stimulated in patients treated with TPO mimetics. This hypothesis will need to be tested in an appropriate clinical trial to assess the potential benefits of TPO mimetics for the treatment of thrombocytopenia in CLL patients. Disclosures:Loberg:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

The Cellular Classification and Separation of Nucleated Blood Cells by Supravital Cell Staining with Acridine Orange

Abstract 4907Immunohistochemical staining and flow cytometry are performed on the tissue to distinguish specific cells. The detection of diseases is made by the flow cytometry using monoclonal antibodies on peripheral blood (PB) or bone marrow cells. The diagnosis of leukemia is made with PB and marrow cells using antibodies to cluster of differentiation (CD) antigens specific to leukemic cells (e.g.,CD13, CD33). Therefore, conventional approaches to identify cellular phenotypes are being replaced by immunophenotyping using flow cytometry. However antibodies satisfying for needs are not always available. In this case, the generation of such antibody to each specific antigen also causes problems of producing the respective recombinant antigen for immunization. When attempting to construct new animal models using other than mice and humans, this issue becomes one of the serious limiting factors in developing research. In addition, the opportunity is increasing to classify unexperienced types of cells such as cells derived from iPS cells and others. We therefore consider that it is important to develop methods to classify and separate specific cells of interest without antibodies. As supravital cell staining with acridine orange (AO) is introduced in 1960's (Jacson JF, Blood, 1961; Lewis M. et al. Blood, 1962), this metachromatic fluorescent dye rapidly stains DNA and RNA independently. Morphological abnormalities of human erythrocytes such as red cell fragment and large platelets are detectable (Nagai Y et al., Int. J Lab Hematol., 2008). AO emits green fluorescence when it binds to the double-stranded DNA and also red fluorescence when it binds to the single-stranded RNA. Consequently flow cytometry of cells stained with AO is suitable for analyzing blood cells in four parameter, which are scattered light intensity (FSC and SSC) and fluorescence intensity (DNA content and RNA content). This analysis method has the possibility of developing the classifying and separating method for abnormalities.Xenopus laevis(X. laevis) has various nucleated blood cells, which are erythrocytes, leukocytes, and thrombocytes, and their progenitors that are not classified yet. As the first step to identify cells with increased RNA content, we chose X. laevis blood cells as a new model of AO staining for flow cytometry by FACS Aria II cell sorter based on the content of DNA and RNA. Since collected cells were stained by May-Griinwald-Giemsa on cytocentrifuge preparations. The population in lower content of DNA and RNA is composed of erythrocytes (95.7±1.3% of whole PB cells). The population in higher concentration of DNA and RNA was pure leukocytes fraction (0.7±0.6% of whole PB cells) expressing the mRNA of X. laevis myeloperoxidase. This population was then fractionated with the higher content of RNA containing eosinophils and basophils (48.7±29.9%), and lower content of RNA contained neutrophils (50.9±29.9%). The proportion of the peripheral thrombocytes in lower forward light scatter (FSC) and side light scatter (SSC) ranges was 0.8±0.8% of whole PB cells, and the expression of c-Mpl, CD41 and Fli-1 mRNA was detected in the sorted cells by RT-PCR. Furthermore, sorted cells were confirmed by immunohistochemical staining by anti X. laevis thrombocyte monoclonal antibody. To reveal the characteristics of the abnormal PB cells, we compared the normal PB with the PB of phenylhydrazine (PHZ) induced anemic X. laevis. At 8 days after treated with PHZ, immature hematopoietic cells were appeared in circulating blood. We isolated these cells with high content of DNA and RNA, and the population in lower FSC and higher SSC with crossover analysis of light emission by AO, suggesting that this method could identify abnormalities in whole PB. The character of these cells was morphologically small and high nuclear cytoplasmic ratio. We also applied this method to blood cells of Rana catesbeiana, American bull frog. Despite the differences in size of erythrocytes, thrombocytes and leukocytes, Rana catesbeiana PB was similarly sorted to X. laevis PB by cellular DNA/RNA content. These results suggest that this method can sort nucleated cells in various species by crossover analysis of light emission by AO. Our study showed that this method has the advantage to characterize of human leukemia cells, tumor cells, iPS-derived cells and nucleated blood cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Megakaryocytes Do Not Express Erythropoietin Receptor (EpoR) and Do Not Respond to Recombinant Human Erythropoietin (rHuEpo) Stimulation

Abstract 4783Recently, clinical studies were conducted in chronic kidney disease of type 2 diabetes patients that suggested an increased risk of stroke in patients receiving Erythropoiesis Stimulating Agents (ESAs) compared to placebo. One proposed explanation is that megakaryocytes and/or platelets express EpoR and respond to ESA challenge. We evaluated the underlying and untested assumption that megakaryocytes or platelets express a functional EpoR using flow cytometry assays developed to detect cell surface EpoR using validated EpoR-specific antibodies. Additionally, in order to investigate whether megakaryocytes/platelets are responsive to ESAs, the activation status of downstream signaling proteins was evaluated after stimulation with ESAs or vehicle control.Whole bone marrow aspirates were collected from healthy volunteers and bone marrow mononuclear cells (BMMCs) isolated by Ficoll separation (n=25). Megakaryocytes were isolated from BMMCs by using CD61 immuno-magnetic bead capture and analyzed by flow cytometry, gating for megakaryocytes using viability, CD41a expression and polyploidy. Platelets were analyzed in peripheral blood from healthy volunteers (n=10) using CD41a as marker.To measure EpoR expression an EpoR-specific monoclonal antibody was used for flow cytometry analysis in both platelets and megakaryocytes that were isolated and gated as described above. Megakaryocytes and platelets were stimulated with rHuEpo (1, 10, and 300 U/mL for 5 and 30 min) and induction of pSTAT5 was measured by intracellular flow cytometry using phospho-specific antibodies. In addition, platelets (CD41a+) were assessed for EpoR expression and functional response to recombinant human erythropoietin (rHuEpo) challenge. Stimulation with thrombopoietin (TPO) was used as a positive control for pSTAT5 induction in megakaryocytes and platelets.While robust EpoR expression was, as expected, observed consistently in UT7/Epo control cells and absent in CMK negative control cells (fold over control – UT7/Epo 12.78 ± 5.03; CMK 0.77 ± 0.22 mean ± 95%CI), no significant expression of EpoR was observed on CD41a+/polyploid megakaryocytes (fold over control − 1.006 ± 0.03; mean ± 95%CI). No activation of downstream signaling (pSTAT5) was detected in megakaryocytes in any of the samples stimulated with rHuEpo at any of the concentrations or timepoints analyzed, while robust pSTAT5 was detected in the UT7/Epo control cell lines. CMK cells were included as a negative control and demonstrated an absence of pSTAT5 induction in response to rHuEpo while a robust induction to TPO was observed (fold over control – UT7/Epo 12.11 ± 7.86; CMK 1.05 ± 0.03; mean ± 95%CI). Similarly, no significant EpoR expression was detected on CD41a+ platelets while the control, c-Mpl expression, was significant (fold over control – EpoR 1.11 ± 0.019; c-Mpl 2.936 ± 0.71; mean ± 95%CI) and no activation of downstream signaling (pSTAT5) was detected in platelets in any of the samples stimulated with rHuEpo at all concentrations and timepoints analyzed. The lack of EpoR expression on megakaryocytes was confirmed by laser scanning cytometric analysis in patient samples.A robust/sensitive platform was developed to profile biologically relevant EpoR expression and function in megakaryocytes and platelets. The assays for EpoR expression and function were sufficiently sensitive to allow characterization during in-vitro differentiation of erythroid progenitors. Using this platform we demonstrated a lack of EpoR expression and functional response to rHuEpo on megakaryocytes and platelets isolated from human bone marrow (CD41a+/CD61+/polyploid). Disclosures:Loberg:Amgen: Employment, Equity Ownership. Rossi:Amgen: Employment, Equity Ownership. Manoukian:Amgen: Employment, Equity Ownership. Wang:Amgen: Employment, Equity Ownership. Paweletz:Amgen: Employment, Equity Ownership. Sable:Amgen: Employment, Equity Ownership. Tudor:Amgen: Employment, Equity Ownership. Patterson:Amgen: Employment, Equity Ownership. McCaffery:Amgen: Employment, Equity Ownership.

Investigation of thrombopoietin (TPO) function in primary chronic lymphocytic leukemia cells.

6613 Background: Immune thrombocytopenia purpura (ITP) is associated with poor prognosis of chronic lymphoid leukemia (CLL). TPO mimetics have been approved for the treatment of chronic ITP and have been suggested for the use in CLL-related ITP. Before testing this hypothesis in the clinic, the hypothesized role of TPO function in CLL cells was investigated. TPO mimetics stimulate platelet production via activation of the TPO receptor, c-Mpl, thus we analyzed c-Mpl cell surface expression and functional response to TPO in primary CLL cells in samples collected from early and late stage CLL patients. Methods: Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. CLL cells were analyzed by flow cytometry and gated based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a tested and validated novel c-Mpl-specific monoclonal antibody by flow cytometry. CLL samples were stimulated with TPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured by intracellular flow cytometry. Platelets (CD41a+) provided internal controls for c-Mpl expression and response to TPO and phorbol myristate acetate was used as a control stimulation to demonstrate that CLL cells were responsive. Results: Analysis of expression and function of c-Mpl was performed on CLL samples (n=89) including 14 patients with thrombocytopenia. While robust c-Mpl expression was observed consistently in platelets from normal and CLL patients (normal: 31.90 ± 2.83, CLL: 26.76 ± 3.31; mean ± SEM), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (1.06 ± 0.01; mean ± SEM). No activation of downstream signaling (pSTAT5) was detected in CLL cells in any of the samples stimulated with TPO (normal: 0.90 ± 0.007, CLL: 1.04 ± 0.02; mean ± SEM), while robust pSTAT5 was detected in platelets (normal: 10.22 ± 0.29, CLL: 6.21 ± 0.23; mean ± SEM). Conclusions: A robust/sensitive platform was developed to profile biologically relevant c-Mpl expression and function in platelets. Using this platform we demonstrate a lack of c-Mpl expression and functional response to TPO challenge in CLL cells suggesting that CLL cells are unlikely to be activated in patients treated with TPO mimetics.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

SummaryHematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes.

The importance of cytogenetics in polycythemia vera, primary myelofibrosis and essential thrombocythemia

The importance of cytogenetics in polycythemia vera, primary myelofibrosis and essential thrombocythemia

The Expression of Thrombopoietin and its Receptor During Different Physiological Stages in the Bovine Ovary

Thrombopoietin (TPO) is known to be involved in megakaryocytopoiesis, but its role in the control of ovarian function is unknown in cattle. The aims of this study were to demonstrate the expression of TPO and its receptor (c-MPL) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy--and to demonstrate that TPO/c-MPL system is expressed clearly in bovine follicles. Real-time RT-PCR (qPCR) and ELISA were applied to investigate mRNA expression of examined factors and TPO protein, respectively. In this investigation, increases in the concentrations of TPO protein and the mRNA expression of TPO and c-MPL were noticed during both early luteal stage and late luteal stage of the oestrous cycle. Furthermore, the expression of TPO/c-MPL system does not show any significant regulation in the CL throughout pregnancy. Highest co-expression of TPO/c-MPL system in both theca interna (TI) and granulosa cells (GC) in small follicles (<10 mm in diameter) was observed in this study that may suggest the possible role of TPO/c-MPL system in proliferation of TI and GC cells. To conclude, the results demonstrate the possible involvement of locally produced TPO/c-MPL system as a 'physiological filter' in bovine ovary where they may promote cell selection by inducing proliferation of viable cells and scavenging non-viable cells and thereby may play an important role in modulation of ovarian function.