Rozprawy doktorskie na temat „Megakaryopoiesis”

Les mégacaryocytes sont les précurseurs cellulaires hautement spécialisés qui produisent des plaquettes via des extensions cytoplasmiques appelées proplaquettes. La formation des proplaquettes exige de profonds changements dans l’organisation du cytosquelette: microtubules et actine. Les formines sont une famille de protéines hautement conservées chez les eucaryotes composées de plusieurs domaines qui régulent le remodelage et la dynamique du cytosquelette d'actine et des microtubules. La plupart des formines sont des effecteurs protéiques des Rho-GTPase. DIAPH1, un membre de la famille des formines, est un homologue chez les mammifères du gène diaphanous de la drosophile qui fonctionne comme un effecteur de la petite GTPase Rho et régule le cytosquelette d'actomyosine ainsi que les microtubules. Il contient le domaine de liaison à Rho (Rho-binding domain) dans la partie amino-terminale et deux régions distinctes d’homologie aux formines, FH1 localisée au centre de la protéine et FH2 dans la partie carboxy-terminale. DIAPH1 co-régule le cytosquelette des microtubules et d'actine à travers respectivement ses régions de FH2 et FH1. DIAPH1 est donc un gène candidat idéal dans toutes les fonctions cellulaires qui exigent une coopération entre cytosquelettes d’actine et de microtubules.L'objectif de ce projet de thèse était d’étudier le rôle de DIAPH1 dans la mégacaryopoïèse. A la fin de la maturation des mégacaryocytes, la formation de proplquettes et la migration sont associées à des modifications importantes de la structure du cytosquelette. Nous avons émis l’hypothèse que grâce à la sa double fonction dans la polymérisation de l'actine et la stabilisation des microtubules, DIAPH1 pourrait jouer un rôle essentiel dans les temps terminaux de la différenciation mégacaryocytaire.Nos résultats ont montré qu’au cours de la différenciation mégacaryocytaire, l’expression de DIAPH1 augmente, alors que celles de DIAPH2 et DIAPH3 diminuent, ce qui suggère que DIAPH1 pourrait jouer un rôle plus important que DIAPH2 et DIAPH3 dans les stades tardifs de la différenciation mégacaryocytaire. Les études en immunomarquage montrent que DIAPH1 co-localise avec l’actine F, la tubuline et la myosine IIa en niveau de la membrane plasmique et des proplaquettes. Nous avons étudié la fonction de DIAPH1 par des stratégies d’invalidation (knockdown) et de surexpression d’une forme active de DIAPH1. Les résultats montrent que DIAPH1 est un effecteur important de Rho, pour réguler négativement la formation des proplaquettes en remodelant le cytosquelette d’actine et les microtubules. Le travail antérieur de notre équipe avait montré que Rho-ROCK régulait aussi négativement la formation des proplaquettes, en inhibant l’activation de la myosine IIa. En inhibant simultanément DIAPH1 et ROCK/myosine, nous avons montré que ces deux voies jouent un rôle additif dans la formation des proplaquettes.Ces résultats suggèrent que la coopération entre les voies DIAPH1 et ROCK/myosine est nécessaire pour la formation de structures cellulaire dépendant de l'actomyosine, telles les fibres de stress et l'anneau contractile en agissant à la fois sur le remodelage du cytosquelette et en assurant un équilibre entre l'actomyosine et microtubules
Megakaryocytes (MKs) are the highly specialized precursor cells that produce platelets via cytoplasm extensions called proplatelets. Proplatelet formation (PPF) requires profound changes in microtubule and actin organization. Formins are a family of highly conserved eukaryotic proteins with multidomains that govern dynamic remodeling of the actin and microtubule cytoskeletons. Most formins are Rho-GTPase effectors proteins. DIAPH1, a member of the formin family, is a mammalian homolog of Drosophila diaphanous gene that works as an effector of the small GTPase Rho and regulates the actomyosin cytoskeleton as well as microtubules. It contains the Rho-binding domain in the N-terminal and two distinct regions of formin homology, FH1 in the center and FH2 in the C-terminus. DIAPH coordinates microtubules and actin cytoskeleton through its FH2 and FH1 regions respectively, making DIAPH an ideal candidate in cell functions that depend closely on the cooperation between the actin and microtubule cytoskeletons.The objective of the project was to decipher the role of DIAPH1 in megakaryopoiesis. At the end of the MK maturation, PPF and MK migration are associated with profound changes in cytoskeleton organization. Due to its dual function in actin polymerization and microtubule stabilization, DIAPH1 was an obvious candidate to play an essential role in PPF and MK migration.Our results showed that DIAPH1 expression increased during MK differentiation, whereas DIAPH2 and DIAPH3 expression decreased, suggesting that DIAPH1 may play a more important role than DIAPH2 and DIAPH3 in the late stages of MK differentiation. Immunostaining showed that DIAPH1 co-localized with F-actin, tubulin and myosin IIa along the plasma membrane and proplatelet. Using a knockdown strategy with shRNA and expression of an active form of DIAPH1, we showed that DIAPH1 is an important effector of Rho that negatively regulates PPF by remodeling actin and microtubule cytoskeletons. A previous work of our team has shown that Rho-ROCK also negatively regulates in PPF by inhibiting myosin IIa activation. By the double inhibition of the DIAPH1 and the ROCK/Myosin pathway, we showed that DIAPH1 and ROCK played additive roles in the negative regulation of PPF. These observations suggest that the cooperation between DIAPH1 and ROCK is required for the formation of cell structures dependent on actomyosin, such as the stress fibers and the contractile ring. Collectively, these results strongly suggest that cooperation of DIAPH1/microtubules and ROCK/Myosin may regulate PPF by modifying the balance between actomyosin and microtubules

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

The immune adaptor ADAP possesses versatile roles in a variety of immune cells, including T cells, dendritic cells, macrophages, and platelets, etc. The most extensivelystudied role of ADAP is that it couples TCR activation to integrin activation and T-cell adhesion. However, the regulation of this adaptor during integrin activation and T-cell adhesion remains unclear. Meantime, the functions of ADAP linked to other immune cells are largely unknown. Work in this thesis have identified Ubc9, the sole SUMO E2 conjugase, as an essential regulator of ADAP in T-cell adhesion. We show that ADAP interacted directly with Ubc9 in vitro and in vivo, and the association was further strengthened in response to anti-CD3 stimulation. The Ubc9 binding domain on ADAP was mapped to a nuclear localisation sequence (aa 674-700) within ADAP. Knockdown of Ubc9 by shRNA or expression of the Ubc9-binding-deficient ADAP mutant significantly decreased TCRinduced integrin adhesion to ICAM-1 and fibronectin, as well as LFA-1 clustering, while having little effect on the TCR proximal signalling responses and TCR-induced IL-2 transcription. Furthermore, downregulation of Ubc9 impaired TCR-mediated Rac1 activation and attenuated the membrane targeting of Rap1 but not RIAM. Taken together, our data demonstrate for the first time that ADAP forms a functional interplay with Ubc9 and Ubc9 plays a selective role in integrin-mediated T-cell adhesion via modulation of Rap1 membrane recruitment and Rac1 activation. Another important finding of this thesis is the identification of a negative regulatory role for ADAP in the megakaryopoiesis. Here we show that in the bone marrow and spleen of ADAP-/- mice, a significant increase in the number of megakaryocytes were observed, and the ADAP-deficient megakaryocytes exhibited potentiated capacity in differentiation and development compared to the WT megakaryocytes. Mechanistically, ADAP directly interacted with STAT1, an indispensable modulator in megakaryopoiesis. Analysis on the activation of STAT1 showed that depletion of ADAP resulted in potentiated STAT1 phosphorylation and transcriptional activity, as well as upregulations of STAT1-regulatory genes. Collectively, these results suggest a novel role of ADAP in megakaryocytes, where ADAP attenuates megakaryopoiesis by direct interaction with STAT1 and negatively modulates the STAT1 activities. In summary, the work in this thesis have illustrated the diverse roles of ADAP in TCR-mediated integrin activation and megakaryopoiesis, and altogether contributed to our current knowledge of the many facets of ADAP in immunity.

Le processus qui aboutit à la formation de plaquettes est appelé mégacaryopoïèse. Les mégacaryocytes (MK) sont de grandes cellules de la moelle osseuse qui par fragmentation dans la circulation sanguine produisent des plaquettes. La régulation extrinsèque ou intrinsèque de ce processus a été largement étudiée. Cependant la régulation épigénétique reste mal connue bien que de nombreuses mutations dans des gènes de régulateurs épigénétiques soient retrouvées dans les hémopathies malignes de la lignée MK. En particulier des mutations du gène de la méthyltransférase EZH2, composant catalytique du Polycomb Repressive Complex 2 (PRC2) ont été détectées dans plusieurs types d’hémopathies. Ces mutations sont soit gain soit perte de fonction suggérant qu’EZH2 peut être à la fois un oncogène ou un gène suppresseur de tumeur. Dans les TE (Thrombocythémie Essentielle) et les MFP (Myélofibrose Primaire), deux néoplasmes myéloprolifératifs (NMPs), qui affectent principalement la lignée MK, des mutations d’EZH2 perte de fonction ont été retrouvées ainsi que dans les DS-AMKL (Down syndrome acute megakaryoblastic leukemia). Cela suggère qu’EZH2 joue un rôle important dans la mégacaryopoïèse normale. La caractérisation de cette fonction pourrait être utile pour mieux appréhender le rôle des mutations d’EZH2 dans les pathologies malignes mégacaryocytaires. Cette thèse peut être divisée en deux parties : 1) Caractérisation du rôle joué par EZH2 dans la mégacaryopoïèse normale et pathologique 2) Développement d‘un outil permettant d’étudier la coopération entre mutations dans les DS-AMKL.1) Lors des temps précoces de la différenciation in vitro des cellules CD34+ de sang de cordon vers la lignée mégacaryocytaire l’inhibition d’EZH2 entraîne l’acquisition plus rapide des marqueurs MK de surface (CD41 et CD42) pour un nombre de mitoses égal. Ceci suggère qu’EZH2 régule la spécification MK des progéniteurs hématopoïétiques. Plus tard dans la différenciation, l'inhibition constante d’EZH2 via des inhibiteurs ou des shRNA, arrête la prolifération et diminue le niveau de ploïdie des MKs en arrêtant la réplication de l’ADN. Ceci est du à la surexpression de plusieurs CDKi (Cyclin dependent kinase inhibiteurs), dont CDKN2D. L'analyse par Chip-Seq a montré que la transcription de CDKN2D est régulée par H3K27me3 au niveau de son promoteur et donc que CDKN2D est une nouvelle cible de PRC2. Dans les MKs les plus matures, l’inhibition d’EZH2 diminue la formation des proplaquettes, ceci est corrélé à des modifications d’expression de gènes régulant le cytosquelette d’actine. L’ensemble de ces résultats a été confirmé sur des MKs de patients porteurs de la mutation JAK2V617F.2) Par la technique CRISPR-Cas9, nous avons introduit dans des iPSC (induced pluripotent stem cells) disomiques et trisomiques pour le chromosome 21, la mutation GATA1s présente chez tous les patients avec une DS-AMKL. Nous avons montré que ces mutations modifiaient le cadre de lecture dans l’exon 2 et entrainaient l’expression de la forme courte de GATA1 (GATA1s). Nous sommes en train d'effectuer des études fonctionnelles ainsi que d’introduire d’autres mutations, y compris celles d’EZH2 pour modéliser la maladie.Au cours de cette thèse nous avons montré que l’inhibition d’EZH2 régule les temps initiaux de la mégacaryopoïèse en accélérant la spécification cellulaire au niveau des progéniteurs et ensuite la maturation terminale en inhibant profondément la polyploïdisation par surexpression de plusieurs CDKi dont CDKN2D et en inhibant la formation des plaquettes par un effet sur le cytosquelette d’actine. Ces résultats pourront être utiles pour mieux comprendre le rôle de la perte de fonction d’EZH2 dans les hémopathies malignes de la lignée mégacaryocytaire
The process that leads to platelet production is called megakaryopoiesis. Megakaryocytes (MK) are the large bone marrow cells that produce platelets by fragmentation in the blood flow. The extrinsic and intrinsic regulation of megakaryopoiesis has been largely studied. However, the epigenetic regulation remains poorly known although numerous mutations in genes of epigenetic regulators have been found in patients with MK hematological malignancies. The methyltransferase EZH2, the catalytic component of Polycomb Repressive Complex 2 (PRC2) is among the most studied epigenetic regulators. EZH2 is also mutated in many malignant hematological disorders where it can be an oncogene or a tumor suppressor gene. Particularly in ET (Essential Thrombocythemia) and PMF (Primary Myelofibrosis), two myeloproliferative neoplasms (MPNs) that affect mainly the MK lineage, loss of function EZH2 mutations have been found as well as in DS-AMKL (Down syndrome acute megakaryoblastic leukemia)Altogether these observations suggest that EZH2 controls normal megakaryopoiesis and characterization of this function could be helpful to understand the role of EZH2 in MK malignant diseases.This thesis can be divided in two parts:1) Characterization of the role of EZH2 in normal and pathological megakaryopoiesis 2) Establishment of a cellular tool to study the cooperation between the different mutations of DS-AMKL. RESULTS1) Using CD34+ cells isolated from cord blood, we showed that at early stages of differentiation, EZH2 inhibition accelerates the acquisition of MK surface markers (CD41a and CD42a) without increasing proliferation suggesting that EZH2 regulates the specification towards the MK lineage. Later in differentiation the constant inhibition of EZH2 via inhibitors or shRNAs, produced a proliferation arrest and a decrease in ploidy level that was related to an arrest in DNA replication due to an upregulation of several CDKi (Cyclin dependent kinase inhibitors), more particularly CDKN2D. Chip-Seq analysis demonstrated that CDKN2D is effectively regulated by H3K27me3 and is a new target of PRC2. This inhibition of ploidization by EZH2 inhibition was confirmed in MK from JAK2V617F patients. Furthermore in the more mature MKs (normal or JAK2V617F) we observed a defect in proplatelet formation, which was associated with an abnormal expression of genes regulating the actin filament. 2) By CRISPR-Cas 9, in iPSCs either disomic or chromosome 21 trisomic, we introduced, the GATA1s mutation present in all DS-AMKL patients. We confirmed at the gene and protein level that this genome editing has been correctly performed and that it induces as previously observed a blockage in erythroid differentiation. We are now carrying out the complete functional characterization together with the introduction of other mutations of DS-AMKL including EZH2.CONCLUSIONThis study describes EZH2 as a regulator of megakaryopoiesis via an initial control of cell specification and then of MK maturation. These results will be useful to better understand the role that EZH2 plays in diseases affecting the MK lineage such as MPNs and DS-AMKL

Investigations on platelet-derived growth factor (PDGF) and serotonin (5-HT), molecules stored in platelet granules, imply their potential effects in regulating megakaryopoiesis, which also intimates the existence of an autocrine and/or paracrine loop constructed by megakaryocytes/platelets and their granular constituents. In addition, numerous reports indicate that melatonin, a derivative from serotonin effectively enhances platelet counts in patients with thrombocytopenia. However, their exact roles on human megakaryocytes and the underlying mechanisms remain unknown. Present studies showed that PDGF, like thrombopoietin (TPO), significantly promoted platelet recovery and the formation of bone marrow colony-forming unit-megakaryocyte (CFU-MK) in an irradiated-mouse model. An increased number of hematopoietic stem/progenitor cells and a reduction of apoptosis were found in the bone marrow aspirate. In the M-07e apoptotic model, PDGF had a similar anti-apoptotic effect as TPO on megakaryocytes. Our findings demonstrated that PDGF activated the PI3-k/Akt signaling pathway, while addition of imatinib mesylate reduced p-Akt expression. Our findings suggested that the PDGF-initiated radioprotective effect is likely to be mediated via PDGF receptors (PDGFRs) with subsequent activation of the PI3-k/Akt pathway. We also provide a possible explanation that blockade of PDGFR may reduce thrombopoiesis and play a role in imatinib mesylate-induced thrombocytopenia. We explored how serotonin regulated megakaryopoiesis and proplatelet formation. Our results indicated that serotonin (5-HT) significantly promoted CFU-MK formation and reduced apoptosis on megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5-HT2 receptor inhibitor. In addition, serotonin was able to stimulate the F-actin reorganization in megakaryocytes through activating the p-Erk1/2 expression. Bone marrow mesenchymal stromal cells (MSCs) are important in regulating megakaryopoiesis through stimulating the release of thrombopoietic growth factor, such as TPO. Our studies suggested that when activated by serotonin, bone marrow MSCs were induced to release significant amount of TPO. Furthermore, thousands of membrane-derived microparticles (MPs) arose from MSCs and the TPO RNA/proteins contained within MPs were also considerably increased under serotonin treatment. In summary, our findings demonstrated an important role serotonin played on megakaryopoiesis. This effect was likely mediated via 5HT2 receptors with subsequent activation of Akt and Erk 1/2 phosphorylation, which led to survival of megakaryocytes and proplatelet formation. Serotonin also stimulated TPO released from MSCs in both dissociative and MP-encapsulated form, which indirectly promoted megakaryopoiesis. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells. The effects of melatonin on megakaryopoiesis were also determined in our studies. Our findings showed that melatonin enhanced proliferation and reduced doxorubicin-induced toxicity on MKs. We further demonstrated the mechanism for melatonin-mediated protection on MKs maybe via repair of G2/M phase cell cycle arrest and inhibition of cell apoptosis on MK cells.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Les néoplasmes myéloprolifératifs (NMPs) classiques BCR-ABL négatifs regroupent la Polyglobulie de Vaquez, la Thrombocytémie Essentielle et la Myélofibrose Primaire. Ce sont des pathologies malignes clonales entraînées par la signalisation constitutive de la voie JAK2/STAT en raison de mutations somatiques acquises qui affectent trois gènes, JAK2, CALR et MPL. Il s’agit des mutations “motrices” de la maladie responsable du syndrome myéloprolifératif et du phénotype. Cependant CALR n’est pas une molécule de signalisation mais une chaperonne du réticulum endoplasmique. En utilisant des lignées dépendantes de facteurs de croissance soit murines (Ba/F3) soit humaines (UT-7), des cellules primaires de patients et des modèles murins nous avons montré que les mutants CALRdel52 et CALRins5 avaient acquis de nouvelles propriétés qui en font des molécules de signalisation en induisant: - une indépendance aux facteurs de croissance uniquement lorsque MPL, le récepteur de la thrombopoïétine est exprimé ; - une phosphorylation constitutive de JAK2, des STAT1, 3 et 5 et une activation faible des voies PI3K/AKT et ERK1/2, suggérant une activation de MPL/JAK2 par les mutants CALR différente de celle induite par JAK2V617F. De manière intéressante un mutant de la CALR ayant une délétion entière de l’exon 9 n’est pas transformant, suggérant que l’activité oncogénique est liée à la présence de la nouvelle séquence C-terminale. L’activation de JAK2 uniquement par MPL en présence des mutants CALR pourrait expliquer le phénotype mégacaryocytaire/plaquettaire de ces NMPs. Cette activation de MPL au contraire de celle exercée par JAK2V617F a lieu non seulement à la membrane mais aussi dans le cytoplasme.Les modèles murins ont montré que les mutants CALR étaient responsables de la maladie et que celle-ci était dépendante de MPL, validant les résultats obtenus sur les lignées.Nous avons également montré que contrairement à JAK2V617F, les mutants de la CALR induisent chez l’homme une dominance clonale très tôt au niveau du compartiment des cellules souches. L’ensemble de ces résultats contribue à une meilleure compréhension du rôle des mutations CALR dans les NMPs. La démonstration que les molécules mutées sont présentes à la surface cellulaire ouvre la voie à des immunothérapies ciblant le nouveau peptide C-terminal
Classical BCR-ABL negative myeloproliferative neoplasms (MPNs) include three disorders: Polycythemia Vera, Essential Thrombocythemia and Primary Myelofibrosis. They are clonal malignant diseases driven by the constitutive JAK2/STAT signaling pathway due to acquired somatic mutations affecting three genes: JAK2, CALR and MPL. These are the "driver" mutations of the disease responsible of the myeloproliferation and of the disease phenotype. However, CALR is not a signaling molecule, but a chaperonne of the endoplasmic reticulum. Using murine (Ba/F3) and human (UT-7) cell lines dependent on growth factors and primary patient cells and mouse model, we have shown that the CALRdel52 and CALRins5 mutants have acquired new signaling properties and induce:- growth factor independence only when MPL, the thrombopoietin receptor, is expressed;- constitutive phosphorylation of JAK2, of STAT1, 3 and 5 and a low activation of the PI3K/AKT and ERK1/2 pathways, suggesting an activation of MPL/JAK2 by a different manner than JAK2V617F. Interestingly, a CALR mutant deleted for the entire exon 9 has not transformation properties suggesting that the oncogenic activity is related to the presence of the new C-terminal sequence. This JAK2 activation only by MPL in presence of CALR mutants could explain the megakaryocytic/platelet phenotype of these MPNs.The use of a mouse modeling using retroviral vectors and bone marrow transplantation has shown that CALRdel52 and ins5 were really the drivers of the disease and that in vivo the thrombocytosis was dependent of MPL validating the results obtained in vitro.In addition, we have shown that in human, CALR mutants induce a clonal dominance early in the stem cell compartment in ET. This is in sharp contrast with JAK2V617F in ET. Overall, these results contribute to a better comprehension of the role of CALR mutations in MPNs. Furthermore, the demonstration that the CALR mutants are expressed at the cell surface open the way to the development of new immunotherapy targetting the new C-terminus peptide

The aim of this project was to investigate the transcriptome of human haematopoietic stem cells (HSCs), megakaryocytes and platelets to gain insights into steady state and accelerated thrombopoiesis that occurs in states of haemostatic demand and in thrombosis by applying these findings to the pathological setting of acute coronary thrombosis. To investigate transcriptional heterogeneity within the human HSC population, single cell RNA sequencing was performed in human bone marrow HSCs. Transcriptionally distinct subpopulations were identified including two megakaryocyte biased subsets with potentially differing functional relevance. Both populations expressed megakaryocyte specific transcripts, one of which also co-expressed common myeloid and megakaryocyte-erythroid progenitor transcripts while the other did not. This study represents the first interrogation of the human bone marrow megakaryocyte transcriptome. Cells were collected from healthy human bone marrow and analysed by low input and single cell RNA sequencing. To identify novel drivers of megakaryocyte maturation, the human bone marrow megakaryocyte transcriptome was compared to that of megakaryocytes cultured from human CD34+ cells, a process known to generate immature megakaryocytes. Transcriptional signatures associated with increasing megakaryocyte ploidy were then investigated. Increasing megakaryocyte ploidy level was found to be associated with an upregulation of transcripts involved in translation and protein processing as well as expression of a number of transmembrane receptors which might have functional relevance. Finally, the pathological setting of acute coronary thrombosis was used as a model for accelerated thrombopoiesis. Megakaryocyte and platelet transcriptomes were compared between patients with acute myocardial infarction (AMI) as well as severe coronary disease and a control group. The transcriptional signature relating to disease compared to control in megakaryocytes included upregulation of platelet activation related transcripts in megakaryocytes isolated from patients with AMI and severe coronary artery disease.

Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

Doctor of Philosophy (PhD)
Gene expression is known to be regulated at the level of transcription. Recently, however, there has been a growing realisation of the importance of gene regulation at the post-transcriptional level, namely at the level of pre-mRNA processing (5’ capping, splicing and polyadenylation), nuclear export, mRNA localisation and translation. Erythroid krüppel-like factor (Eklf) is the founding member of the Krüppel-like factor (Klf) family of transcription factors and plays an important role in erythropoiesis. In addition to its nuclear presence, Eklf was recently found to localise to the cytoplasm and this observation prompted us to examine whether this protein has a role as an RNA-binding protein, in addition to its well-characterised DNA-binding function. In this thesis we demonstrate that Eklf displays RNA-binding activity in an in vitro and in vivo context through the use of its classical zinc finger (ZF) domains. Furthermore, using two independent in vitro assays, we show that Eklf has a preference for A and U RNA homoribopolymers. These results represent the first description of RNA-binding by a member of the Klf family. We developed a dominant negative mutant of Eklf by expressing its ZF region in murine erythroleukaemia (MEL) cells. We used this to investigate the importance of this protein in haematopoietic lineage decisions by examining its effect on the multipotent K562 cell line. We provide evidence that Eklf appears to be critical not only for the promotion of erythropoiesis, but also for the inhibition of megakaryopoiesis.

La protéine Bcl-xL fait partie de la famille des protéines anti-apoptotiques Bcl-2. Il a été montré que cette protéine avait un rôle majeur dans la formation des plaquettes chez la souris (mégacaryopoïèse). Une dérégulation de cette protéine pourrait aboutir à une altération de la mégacaryopoïèse et donner des pathologies humaines comme des thrombopénies chroniques. Une des causes de thrombopénies chroniques est le purpura thrombopénique immunologique (ou PTI), qui associe deux mécanismes physiopathologiques : une destruction auto-immune des plaquettes et une insuffisance de leur production par la moelle osseuse. Le PTI est un diagnostic d’exclusion par élimination de toutes les causes connues de thrombopénie. Au sein de notre cohorte de patients suivis en médecine interne pour cette maladie, nous avons identifié certains patients qui présentaient un profil non-immunologique, c’est-à-dire l’absence d’auto-immunité et une non réponse à tous les traitements immunomodulateurs, ou pas d’indication à un traitement compte tenu d’un taux de plaquettes suffisant. Nous montrons dans ce travail de thèse que Bcl-xL est nécessaire pour la survie du mégacaryocyte humain pendant toute la mégacaryopoïèse, à la différence de la souris. Par ailleurs, certains patients ont une altération intrinsèque de la formation des proplaquettes, et certains d’entre eux ont également une diminution de l’ARN messager et de la protéine Bcl-xL dans leurs plaquettes. Ces observations nouvelles suggèrent l’implication de Bcl-xL dans la physiopathologie de leur maladie et ouvrent la voie à l’identification d’une potentielle nouvelle cause de thrombopénie chronique
The Bcl-xL protein is a member of Bcl-2 anti-apoptotic proteins. It has been shown in mouse that this protein had a major role in platelet production (megakaryopoiesis). Bcl-xL deregulation could lead to megakaryopoiesis impairement and explain some human diseases such as chronic thrombocytopenias. One cause of chronic thrombocytopenia is immune thrombocytopenia (ITP) that associates 2 pathophysiological mechanisms: an immune-mediated platelet destruction and an insufficient production from the bone marrow cells. ITP is a diagnosis of exclusion when all known causes of thrombocytopenia have been ruled out by diagnosis work-up. In ITP cohort of patients followed in our internal medicine department, we have identified some patients with a haematological profile of their disease, ie absence of overt features of auto-immunity, and absence of response to immunomudulatory treatments, or no indication to such treatment because of sufficient platelet count. We demonstrate in this study that Bcl-xL is necessary for megakaryocyte survival during all megakaryopoiesis, contrary to what was found in mouse. Moreover, some patients have an intrinsically impaired proplatelet formation, and some of them also have a decrease of Bcl-xL mRNA and protein in their platelets. These novel observations suggest that a deregulation of Bcl-xL is a possible cause of their disease and lead the way to the identification of a potentially new cause of chronic thrombocytopenia in human

La thrombopénie familiale avec prédisposition à la leucémie aiguë myéloïde (FPD/AML) est une pathologie rare caractérisée par une thrombocytopénie. La FPD/AML est causée par des mutations germinales dans le gène codant le facteur de transcription RUNX1. Ces mutations sont de type dominant négatif (DN), associées à un risque plus élevé de développer une leucémie, ou de type haploinsuffisance (HI) induisant une thrombocytopénie seule. Nous avons démontré une diminution presque complète de l’expression du répresseur transcriptionnel ZBTB1 dans les progéniteurs hématopoïétiques des patients porteurs de mutations DN. Le gène ZBTB1 pourrait être une cible directe de RUNX1, et pourrait contribuer à la dérégulation de la lymphopoïèse T conduisant à une prédisposition à la LAL-T.Nous avons identifié dans les cellules lymphocytaires murines le site de fixation de RUNX1 sur un enhanceur localisé à 270 kb en amont du promoteur de Zbtb1, et ce aux stades doubles négatifs pour les marqueurs CD4/CD8. Dans les stades plus matures (CD4+CD48+), cette fixation n’est pas observée. En utilisant des lignées lymphocytaires humaines représentant les différents stades doubles négatifs CD4/CD8 de la différenciation lymphocytaire, nous ne sommes pas arrivés à démontrer cette liaison suggérant qu’elle a lieu à un stade très précis et transitoire difficilement identifiable. Les souris KO Runx1 et KO Zbtb1 montrent un blocage de la lymphopoïèse T dès les stades les plus précoces de la maturation thymique. Nous voulions démontrer que la surexpression de Zbtb1 dans un contexte KO Runx1 aboutirait à un sauvetage du phénotype lymphocytaire. Pour cela, nous avons utilisé un modèle in vitro (culture des progéniteurs hématopoïétiques sur des lobes thymiques) et in vivo reposant sur la greffe de souris irradiées par des progéniteurs hématopoïétiques de souris KO Runx1 surexprimant Zbtb1. Le KO Runx1 incomplet et une quasi-absence de la prise de greffe dans les conditions de KO Runx1 ne nous ont pas permis de valider notre hypothèse. Cependant nous avons pu observer que ZBTB1 régule négativement le compartiment des cellules souches et la prise de greffe.Nous nous sommes aussi intéressés au phénotype mégacaryocytaire des souris KO Zbtb1. De manière intéressante, ces souris montrent in vivo un défaut du cycle cellulaire des mégacaryocytes, tandis que in vitro une diminution drastique de la différenciation mégacaryocytaire est observée suggérant ainsi une compensation du microenvironnement in vivo. De plus, nous avons montré une régulation négative directe de ZBTB1 par RUNX1 dans les mégacaryocytes humains. Dans la deuxième partie de ma thèse nous nous sommes intéressés au mécanisme d’induction de la leucémie chez un patient FPD/AML porteur de mutation de type DN (RUNX1R174Q), nous avons identifié une mutation additionnelle à une fréquence de 1% dans le gène TET2 ayant contribué à l’amplification du clone pré-leucémique. Actuellement nous étudions la coopération entre la mutation de RUNX1R174Q et le shTET2 in vivo en greffant des souris NSG avec des cellules progénitrices humaines CD34+ portant la mutation RUNX1 et le shTET2 qui mime la mutation perte de fonction de TET2P1962T observée chez le patient. Des résultats prometteurs montrent une prise de greffe primaire et secondaire plus importante dans les conditions RUNX1R174Q/shTET2 et shTET2 seul. Les expériences in vitro réalisées en parallèle montrent que la mutation de RUNX1R174Q induit des dommages à l’ADN alors que la diminution de l’expression de TET2 par shARN induit une prolifération augmentée des progéniteurs hématopoïétiques. L’addition des deux mutations pourrait ainsi conduire à l’acquisition de mutations additionnelles et à une transformation leucémique
Familial platelet disorder with predisposition to acute myeloid leukaemia (FPD/AML) is a rare condition characterized by thrombocytopenia. FPD/AML is caused by germline mutations in the gene coding for the transcription factor RUNX1. These mutations are devided on dominant-negative (DN) mutations associated with a higher risk of developing leukaemia or haploinsufficiency (HI) mutations inducing thrombocytopenia alone.We have demonstrated an almost complete decrease in the expression of the transcriptional repressor ZBTB1 in hematopoietic progenitors of patients with DN-type mutations. ZBTB1 could be a direct target of RUNX1, and could contribute to deregulation of T lymphopoiesis, leading to a predisposition to T-ALL.In murine immature T lymphocytes (CD4-CD8- stages), we demonstrated a fixation of RUNX1 on an enhancer at 270 kb upstream of Zbtb1 promoter. This fixation is no longer observed in the more mature stages (CD4+CD8+). Using human lymphocyte cell lines representing the different CD4-CD8- differentiation stages, we have not been able to demonstrate this binding suggesting that it takes place at a very precise and transitory stage that is difficult to identify.The KO Runx1 and KO Zbtb1 mice show a blockade of T lymphopoiesis in the earliest stages of thymic maturation. We wanted to demonstrate that the overexpression of Zbtb1 in a KO Runx1 context would result at least in a partial rescue of the lymphocyte phenotype. For this we used an in vitro model (culture of hematopoietic progenitors on thymic lobes) and in vivo based on the grafting of irradiated mice with hematopoietic progenitors of KO Runx1 overexpressing Zbtb1. The incomplete KO Runx1 and the almost complete absence of engraftment in the KO Runx1 conditions did not allow us to validate our hypothesis. However, we observed that ZBTB1 negatively regulates the stem cell compartment and the engraftment capacity.We also studied the megakaryocytic phenotype of KO Zbtb1 mice. Interestingly, these mice show, in vivo, a megakaryocyte cell cycle defect; while in vitro a drastic decrease in megakaryocytic differentiation is observed suggesting an in vivo micro-environmental compensation. We also showed a direct negative regulation of ZBTB1 by RUNX1 in human megakaryoycytes.In the second part of my thesis, we investigated the mechanism of induction of leukemia in an FPD/AML patient with a DN-type mutation (RUNX1R174Q). We demonstrated an additional mutation at a frequency of 1% in TET2 gene, which contribute to the amplification of a preleucemic clone.Currently we are studying the cooperation between the RUNX1R174Q mutation and the shTET2 in vivo by grafting NSG mice with human CD34+ progenitor cells carrying RUNX1R174Q mutation and an shTET2, which mimics the loss of function of TET2 observed in the patient. Promising results show greater primary and secondary graft under RUNX1R174Q /shTET2 and shTET2 conditions. The in vitro experiments carried out, show that the mutation of RUNX1R174Q induces DNA damages, whereas the decrease in the expression of TET2 by shRNA induces an increased proliferation of hematopoietic progenitors. The addition of the two mutations could thus lead to the acquisition of additional mutations and to a leukemic transformation

Les plaquettes sanguines ont comme rôle principal d’arrêter les saignements. Elles sont produites dans la moelle osseuse par des mégacaryocytes (MK) qui proviennent de la différenciation des cellules souches hématopoïétiques (CSH). L’objectif de ma thèse a été d’identifier les éléments cellulaires du microenvironnement contrôlant la mégacaryopoïèse. Mon travail a permis d’identifier une population particulière de progéniteurs hépatiques du foie foetal capable de promouvoir in vitro les étapes précoces de la mégacaryopoïèse à partir de CSH humaines et murines (Brouard et al., 2017). Le rôle des cellules endothéliales (EC) dans les étapes tardives de maturation a été étudié après purification à partir de moelle humaine dans des expériences de co-culture avec des MK prédifférenciés. Mes résultats montrent que ces EC ont la propriété unique, par comparaison avec des EC d’autres tissus, de promouvoir la maturation des MK. Une analyse transcriptionnelle différentielle a permis d’identifier des effecteurs possibles ouvrant des pistes pour mieux comprendre les mécanismes de la mégacaryopoïèse et pour améliorer la production des plaquettes en culture
The main role of platelets is to stop bleeding. They are produced in the bone marrow by megakaryocytes (MK) that are produced by the differentiation of hematopoietic stem cells (HSC). The objective of my thesis was to identify the cellular elements of the microenvironment controlling megakaryopoiesis. My work has identified a particular population of hepatic progenitors from the fetal liver capable of promoting in vitro the early stages of megakaryopoiesis from human and murine HSC (Brouard et al., 2017). The role of endothelial cells (EC), purified from human bone marrow, in late maturation stages was studied in co-culture experiments with predifferentiated MK. My results show that these EC have the unique property in comparison with EC from other tissues, of promoting the maturation of MK. A differential transcriptional analysis identified possible effectors that could lead to a better understanding of the mechanisms of megakaryopoiesis and improve platelet production in culture

L’objectif de cette thèse est d’étudier, à l’aide de modèles mathématiques, le mécanisme de régulation qui permet au corps de maintenir une quantité optimale de plaquettes sanguines. Le premier chapitre présente le contexte biologique et mathématique. Dans un second chapitre, un modèle pour la mégacaryopoïèse est introduit qui suppose une régulation ponctuelle par le nombre de plaquettes du taux de différentiation des cellules souches vers la lignée mégacaryocytaire et du nombre de plaquettes produites par mégacaryocyte. Nous montrons que la dynamique de ce modèle est régie par une équation différentielle à retard x'(t) = -?x(t)+f(x(t))g(x(t-t)), et nous obtenons ensuite de nouvelles conditions suffisantes pour la stabilité et l’oscillation des solutions de cette équation. Dans le troisième chapitre, nous analysons un second modèle pour la mégacaryopoïèse qui considère cette fois-ci une régulation opérée en continu uniquement via la vitesse de maturation des mégacaryoblastes. L’analyse de stabilité nécessite d’adapter un cadre pré-existant aux cas où le paramètre de bifurcation n’est pas le retard, et permet de montrer que l’augmentation du taux de mort des mégacaryoblastes conduit à l’apparition de solutions périodiques, en accord avec les observations cliniques de la thrombopénie cyclique amégacaryocytaire. Le dernier chapitre est consacré l’analyse de stabilité d’une équation différentielle à deux retards qui apparait notamment dans le cadre de la mégacaryopoïèse lorsque l’on considère que les plaquettes ont une durée de vie limitée
The object of this thesis is the study, using mathematical models, of the regulation mechanism maintaining an optimal quantity of blood platelets. The first chapter presents the biological and mathematical context of the thesis. In a second chapter, we introduce a model for megakaryopoiesis assuming a regulation by the platelet quantity of both the differentiation rate of stem cells to the platelet cell line and the amount of platelets produced by each megakaryocyte. We show that the dynamic of this model corresponds to a delay differential equation x'(t) = -?x(t) + f(x(t))g(x(t - t)), and we obtain for this equation new sufficient conditions for stability and for the oscillation of solutions. In a third chapter, we analyze a second model for megakaryopoiesis in which the regulation is continuous through the maturation speed of megakaryocyte progenitors. The stability analysis requires to adapt a pre-existing framework to problems where the bifurcation parameter is not the delay, and allows to show that increasing the death rate of megakaryocyte progenitors leads to the onset of periodic solutions, in agreement with clinical observation of amegakaryocytic cyclical thrombocytopenia. The last chapter covers a differential equation with two delays that appears among others in a model of platelet production which considers that platelet death can both age-independent and age-dependent

博士
慈濟大學
醫學科學研究所
105
Dengue virus (DENV) causes 50-100 million infections annually worldwide. Thrombocytopenia is the common feature of dengue viruses-induced mild dengue fever, more severe dengue hemorrhage fever, and dengue shock syndrome. Megakaryopoiesis is the differentiation and maturation processes of megakaryocytes which are the precursors of platelets. Previous studies have demonstrated that DENV can induce thrombocytopenia by megakaryopoiesis suppression; however, the mechanism for megakaryopoiesis suppression, such as whether viral replication is required or what molecule of DENV involved is still unclear. DENV envelope protein domain III (DENV-EIII) is responsible for binding to the host cells. This study found DENV-EIII could suppress TPA (12-O-tetradecanoylphorbol-13-acetate)-induced megakaryocytic differentiation of human erythroleukemia cells, cytokines-triggered megakaryocytic differentiation of CD34+ cells derived from human umbilical cord blood, and thrombopoietin-induced megakaryocytic differentiation of mice bone marrow cells. Megakaryopoiesis suppression was also found in the bone marrow of DENV-EIII-injected C57BL/6J mice like DENV-injected mice. Further, this study demonstrated that DENV-EIII-induced megakaryopoiesis suppression is caused by DENV-EIII directly binding to megakaryocytes and triggering autophagy impairment and apoptosis, and is independent of viral replication. In conclusion, DENV-caused thrombocytopenia is mediated by DENV-EIII-elicited megakaryopoiesis suppression through binding to megakaryocytes and inducing autophagy impairment and apoptosis. Therefore, DENV-EIII could be developed as a drug target to ameliorate thrombocytopenia in dengue patients.

博士
慈濟大學
醫學科學研究所
101
Megakaryocytes are the precursor cells of platelets and critical for maintaining coagulant functions. Thrombopoietin (TPO), the ligand for c-mpl, stimulates proliferation of committed megakaryocytic progenitors and induces maturation of megakaryocytes. In the first part of this study, we established the in vitro megakaryocyte differentiation system from embryonic stem cells co-cultured with OP9 stromal cells. OP9 stromal cells, derived from mouse bone marrow, are useful feeder cells for hematopoiesis in ES cell co-culture systems. However, OP9 is a primary cell line that has a short in vitro life span. Herein, we immortalized primary OP9 cells with the E6 and E7 genes from human papillomavirus type 16. The immortalized OP9 stromal cells were able to extend their lifespan beyond 42 passages and retain their ability to induce megakaryocyte differentiation from embryonic stem cells. In the second part of this study we examined the pathogenic effect of Bacillus anthracis lethal toxin (LT) on megakaryopoiesis. Anthrax LT is the major virulence factor of B. anthracis. In our previous study, we investigated the suppression of platelet function that was associated with LT-induced mortality and observed that LT injection reduces platelet counts prior to death in mice. However, the mechanism responsible for this effect remains unclear. LT is known to inactivate cellular-mitogen-activated protein kinase (MEK) pathways. Previous studies have also shown that the MEK1/2-extracellular signal-regulated kinase (ERK) pathway is critical for megakaryocytic differentiation. Therefore, we hypothesize that LT might inhibit the progenitor cells of platelets, and thereby induce thrombocytopenic responses. We employed the human eruthroleukemia (HEL) cell line, human cord blood-derived mononuclear cells, CD34+ cells,and mouse bone marrow mononuclear cells to perform in vitro megakaryocytic differentiation. These results show that LT suppresses megakaryopoiesis by killing the megakaryocytes and inhibiting megakaryocytic differentiation. Pretreatments with TPO to up-regulate megakaryopoiesis considerably reduced LT-mediated mortality in mice. Our data indicate that LT-suppressed megakaryopoiesis is involved in LT-mediated pathogenesis.

In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood. Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation. Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood. Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury
In Säugetieren zirkulieren kernlose Thrombozyten im Blutstrom und sind primär für die Aufrechterhaltung der funktionellen Hämostase verantwortlich. Thrombozyten werden im Knochenmark durch Megakaryozyten gebildet, die sich hauptsächlich in direkter Nähe zu Knochenmarkssinusoiden befinden, um Proplättchen in das Blut freizusetzen. Megakaryo-zyten stammen von hämatopoetischen Stammzellen ab und man glaubt, dass sie während ihres Reifungspro¬zesses von der endostalen in die vaskuläre Nische wandern – ein Prozess, der trotz intensiver Forschung noch nicht vollständig verstanden ist. Langzeit-Zwei-Photonen-Mikroskopie von Megakaryozyten und des Gefäßbaums wurde in murinem Knochenmark von lebenden Tieren in Kombination mit der Analyse der mittleren quadratischen Verschiebung der Zellmigration durchgeführt. Die Megakaryozyten zeigten keine Migration, sondern eine wackelartige Bewegung auf Zeitskalen von 3 Stunden. Die gerichtete Zellmigration führt stets zu einer nicht zufälligen räumlichen Verteilung der Zellen. Daher wurde ein Computermodellierungsalgorithmus entwickelt, der eine zufällige Megakaryo¬zytenverteilung unter Verwendung von realen 3D-Lichtblatt-Fluoreszenzmikroskopie-Datensätzen simuliert. Der direkte Vergleich realer und simuliert zufälliger Megakaryozyten¬verteilungen zeigte, dass MKs stark mit Knochenmarksgefäßen assoziiert sind. Dieses wird jedoch nicht durch Zellmigration verursacht, da nicht-Gefäß-assoziierte MKs zufällig im intervaskulären Raum verteilt waren. Darüber hinaus zeigten Simulationsstudien, dass Megakaryozyten die Migration anderer Zellen im Knochenmark stark beeinträchtigen, da sie als sterische Hindernisse wirken. Es wird angenommen, dass MKs während ihres Reife¬prozesses von den Regionen in der Nähe des Endosteums in Richtung des Gefäßsystems wandern. Die Megakaryozytenverteilung als Funktion ihrer Lokalisierung relativ zu den endo¬stalen Regionen des Knochens wurde durch Lichtblattmikroskopie untersucht. Die Ergebnisse zeigen keine knochenregionabhängige Verteilung von Megakaryozyten. Zusammenge¬nommen widerlegen die neu etablierten Methoden und erzielten Ergebnisse das Modell der Megakaryozyten¬migration während ihrer Reifung. Ischämie-Reperfusionsschaden (I/R) ist eine häufige Komplikation des zerebralen ischämischen Schlaganfalls, bei dem trotz erfolgreicher Rekanalisierung eine Schädigung des Hirngewebes auftritt. Es wurde gezeigt, dass Thrombozyten, Endothelzellen und Immunzellen das Fortschreiten der I/R-Verletzung in experimentellen Mausmodellen 24 Stunden nach der Rekanalisierung beeinflussen. Die zugrundeliegenden Pathomechanismen, insbesondere in den ersten Stunden nach der Rekanalisierung, sind jedoch kaum verstanden. Hier wurden Lichtblattmikroskopie, Zwei-Photonen-Mikroskopie und ergänzende hochkom-plexe Bildanalyse-Workflows zur Untersuchung von Thrombozyten, der Gefäße und Neutro-philen in ischämischen Gehirnen etabliert. Die quantitative Analyse der Thrombusbildung in der ipsilateralen und kontralateralen Hemisphäre zu verschiedenen Zeitpunkten zeigte, dass die Thrombozytenaggregationsbildung während der ersten 8 Stunden nach der Rekanalisierung minimal ist und in beiden Hemisphären auftritt. In Anbetracht dessen, dass zu diesem Zeitpunkt bereits eine maximale Gewebeschädigung vorliegt, kann geschlossen werden, dass die Infarkt¬progression und der neurologische Schaden nicht aus der Bildung von Thrombozytenaggre¬gaten resultieren. Darüber hinaus erlaubte Lichtblattmikroskopie die Neutrophileninfiltration in die infarzierte Hemisphäre zu bestätigen und hier waren die Spiegel des Endothelzellmarkers PECAM1 stark reduziert. Es müssen jedoch weitere Untersuchungen durchgeführt werden, um die Rolle von Neutrophilen und Endothelzellen bei I/R-Verletzungen klar zu identifizieren