Megakaryocyte specification is the process by which discrete hematopoietic subpopulations undergo lineage commitment towards the myeloid compartment, finally specifying as a megakaryocyte erythroid progenitor (MEP) by way of thrombopoietin (TPO) and erythropoietin (EPO) signaling, before becoming a megakaryocyte lineage restricted progenitor that will progressively increase cellular ploidy and compartmentalize its cytoplasm in preparation for platelet production. With the advent of induced pluripotent stem cells (iPSCs), a cell type that is experimentally manipulated to function as embryonically derived pluripotent cells, there now exists the ability to analyze signal transduction throughout discrete phases of hematopoiesis, megakaryocyte lineage cell fate, and platelet production. Recent studies have implicated the aryl hydrocarbon receptor (AHR) as a transcription factor that plays a critical role in multiple aspects of hematopoiesis. These results inspired the hypothesis that AHR signaling may be functionally relevant in the context of megakaryopoiesis. To test this hypothesis, an iPSC directed differentiation strategy was established in order to create a platform upon which to experimentally manipulate AHR signaling throughout megakaryocyte specification. The results demonstrate: 1) iPSC derived hematopoietic progenitor cells (HPCs) undergo exponential expansion upon AHR agonism; 2) AHR antagonism allows for megakaryocyte lineage bias; 3) Optimization of directed-differentiation allows for the examination of AHR signaling in megakaryocyte lineage-restricted cells; 4) AHR signaling suppresses the expression of MPL, the gene that encodes the thrombopoietin receptor (C-MPL) in iPSC derived megakaryocyte lineage committed cells; 5) AHR activation concomitantly suppresses cell surface expression of C-MPL, which may alter the sensitivity of HPCs to TPO signaling; 6) Multiple gene targets are modulated by AHR activation within megakaryocyte lineage cells, providing evidence of a transcriptional program downstream of AHR signaling that preferentially suppresses megakaryocyte specification; 7) A reporter iPSC line of AHR activity provides evidence of endogenous AHR signaling throughout megakaryocyte specification and shows a sharp decline in AHR activity upon megakaryocyte lineage commitment; 8) In a mouse model of megakaryocyte lineage specific AHR knockout, platelet counts are significantly reduced. These data suggest that the AHR plays a significant role in megakaryocyte specification by modulating the expression of multiple lineage specific gene targets, including MPL, the thrombopoietin receptor. / 2017-05-02T00:00:00Z

There exists a need to produce platelets in vitro for use in transfusion medicine, due to increased platelet demands and short shelf life. Our lab uses human induced pluripotent stem cells (iPSCs), as an attractive alternative supply, as iPSCs can be cultured indefinitely and differentiate into almost any cell type. Using a technique called forward programming, we over express three key haematological transcription factors (TFs), pushing iPSCs towards the megakaryocyte lineage, to produce mature megakaryocytes, the platelet precursor cell type. A major limitation of the forward programming technique is a reliance of lentiviral transduction to overexpress the three TFs, which leads to a number of issues including heterogeneity and high experimental costs. To overcome this, I have developed an inducible iPSC line by inserting the forward programming TFs into a genomic safe harbour, using genome editing techniques. TF expression is strictly controlled, with the TFs expressed only after chemical induction. Inducing forward programming is an efficient method for producing mature megakaryocytes and these cells maintain higher purity in long-term cultures, when compared to cells produced by the lentiviral method. Removing the requirement of lentiviral transduction is a major advancement, making forward programming more amenable to scaling-up, thus moving this technology closer towards our goal of producing in vitro platelets for use in transfusion medicine. I have also shown that forward programming generates a bi-potent progenitor population, from which erythroblasts can be generated, by altering only media conditions. As for megakaryocyte cultures, inducing forward programming improves the purity of erythroblasts produced, compared to the lentiviral method. I have developed single cell progenitor assays combined with index sorting of different cell surface markers, to allow retrospective analysis of cells which successfully generate colonies. The aim of this work is to better characterise the progenitor cells produced by forward programming, to allow further study of this cell type. Single cell RNA-seq of megakaryocytes revealed heterogeneity in long-term cultures and also identified novel candidate surface markers that may help to further characterise the progenitor cell population.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22648号 / 医科博第111号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cables1 est impliqué dans la régulation du cycle cellulaire et la survie. Par QPCR et western blot, Cables1 est fortement exprimé dans les cellules souches hématopoïétiques (CSH), les progéniteurs, les cellules de la niche médullaire et les mégakaryocytes. En utilisant un modèle de souris Cables1-/-, nous avons démontré que Cables1 est un régulateur clé de la maintenance homéostatique des CSH àl’état basal et sous stress hématopoïétique. Chez les souris jeunes dépourvues de Cables1, les progéniteurs hématopoïétiques sont hyperprolifératifs et ont un avantage compétitif de repeuplement. La surexpression lentivirale et la déplétion par shRNA de la protéine Cables1 ont respectivement entraîné une régulation positive et négative de p21, indiquant que l'effet de Cables1 sur la prolifération des progéniteurs est partiellement médiée par la régulation de p21. Avec l’âge, les souris déficientes en Cables1 présentent des anomalies du nombre de globules blancs accompagnées d'une réduction significative du compartiment CSH associée à une mobilisation accrue des progéniteurs. De plus, les souris Cables1-/-présentent une sensibilité accrue à un agent myélotoxique à l’irradiation due à des défauts dumicroenvironnement médullaire. Dans les mégacaryocytes, la diminution de Cables1 par shRNA entraîne un défaut de prolifération et unediminution du pourcentage de MK matures. De plus, un défaut de la capacité de formation de proplaquette a été observé après la diminution de Cables1. Ces effets peuvent s’expliquer par une apoptose accrue. En conclusion, Cables1 régule à la fois les progéniteurs et la mégacaryopoïèse. Cables1 donc est essentiel pour l'homéostasie des CSH et le contrôle du stress des CSH. La manipulation del’expression de Cables1 pourrait représenter une opportunité pour optimiser les schémas de chimiothérapie. / Cables1 has been described to be involved in cell cycle regulation and survival. Using QPCR and western blot, we demonstrate for the first time that Cables1 in highly expressed in hematopoietic stem cells, in niche cells and megakaryocytes. Using the Cables1-/- mouse model, we demonstrate that Cables1 is a key regulator of homeostatic HSC maintenance and under hematopoietic stress. Young mice lacking Cables1 showed hyper proliferation within the hematopoietic progenitor and stem cell (HSPC) compartment. Loss ofCables1 conferred increased competitive repopulating capacity to the HSPCs. Lentiviral mediated overexpression and shRNA mediated depletion of Cables1 protein resulted in p21 up and down regulation, respectively, indicating that the effect of Cables1 on HSPC proliferation is partially mediated through regulating p21. By 1,5 to 2 years of age, Cables1 deficient mice displayed anomalies in whiteblood cell counts accompanied by a significant a reduction in the HSC compartment coupled with increased mobilization of HPC. In addition, Cables1-/- mice displayed increased sensitivity to myelotoxic agent and irradiation. These defects are related to abnormal microenvironment. We also investigated Cables1 function during megakaryopoiesis. Down regulation of Cables1 in CD41+CD42- megakaryocytic progenitors resulted in proliferative defect and decreased percentage of mature MKs, which were accompanied by p21(cyclin dependent kinase inhibitor) and Bax (an apoptosis related gene) up-regulation. Moreover, defect of proplatelet forming capacity was observed after Cables1 knockdown, which can also be explained by elevated apoptosis induced by Bax protein. In conclusion, Cables1 regulate both HSPCs and the process of megakaryopoiesis. It represents a opportunities to optimize chemotherapy schemes.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12971号 / 論医科博第2号 / 新制||医科||5(附属図書館) / 32409 / 新制||医科||5 / 横浜市立大学大学院国際総合科学研究科バイオ科学専攻 / (主査)教授 長船 健二, 教授 中畑 龍俊, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Les mécanismes de formation des plaquettes sanguines à partir des mégacaryocytes ne sont pas totalement compris, mais l’environnement médullaire semble y avoir une influence cruciale. Dans ce travail nous montrons que i) les intégrines β3, récepteurs de protéines de matrice extracellulaire, semblent impliquées dans la mégacaryopoïèse et la formation des plaquettes, ii) la différenciation des cellules hématopoïétiques dans un environnement 3D de rigidité comparable à la moelle osseuse améliore la maturation des mégacaryocytes différenciés in vitro et iii) la myosine IIA est impliquée dans la distribution des organelles dans les mégacaryocytes. Parallèlement, Nous avons caractérisé la spécificité d’expression du transgène Pf4-cre pour valider son utilisation dans nos approches expérimentales. Ce travail apporte un éclairage nouveau sur le rôle de la myosine IIA et des intégrines dans les mégacaryocytes et souligne l’influence de la rigidité de l’environnement dans la mégacaryopoïèse. / Megakaryocytes differentiation (megakaryopoiesis) and platelet formation mechanisms are not entirely understood, but the bone marrow environment seems to be crucial in these processes. In this thesis, we show i) that integrin β3, the extracellular matrix protein receptors, are involved in megakaryopoiesis and platelet formation, ii) that recreating a 3D environment of stiffness in the range of that of bone marrow improves the maturation of in vitro differentiated megakaryocytes and iii) a new role for myosin IIA in the cytoplasmic distribution of organelles within the megakaryocyte. As a side-project, we characterized the specificity of expression of the Pf4-cre transgene to validate its use in our experimental approaches. This work enlightens new roles for myosin IIA and integrins in megakaryocytes and indicates that stiffness of the environment influences megakaryopoiesis.

<p>Megakaryocyte cultures are a strong tool for the in vitro investigation of platelet production in platelet disorders. Peripheral blood derived hematopoietic progenitor cells (PB-HPCs) are the most accessible source of HPCs with high potential to produce mature megakaryocytes in vitro; however, they are present in low numbers making peripheral blood an inefficient source. Additionally, a megakaryocyte culture with an optimized thrombopoietin (TPO) concentration is required which can reliably allow the investigation of suppressive effects of antibodies/plasma from immune thrombocytopenia (ITP) patients. In this study, we developed a megakaryocyte culture with the utilization of human PB-HPCs in an efficient fashion resulting in the production of high purity megakaryocytes in a TPO-dependent manner.</p> <p>The mononuclear fraction was collected from 180 mL of peripheral whole blood and CD34+ cells were isolated by a positive selection yielding the average of 5.5 x 105 ± 2.5 x 105 CD34+ cells (n = 18). Using 96-well tissue-culture plates and seeding 10,000 CD34+ cells/well, the average of 13 experiments in triplicate can be set up utilizing isolated CD34+ in an efficient manner. Capitalizing on a TPO dose-dependent megakaryocyte production experiment, 20 ng/mL was established as the TPO concentration which resulted in the production of mature megakaryocytes without reaching the plateau in megakaryopoiesis response. On day 11 of culture, the expression of megakaryocytic lineage (CD41/61+) and maturation (CD41/61+CD42+) markers peaked at 90.65% and 76.10%. In conclusion, this culture system has broad application for investigation of platelet disorders and drug discovery which can be accessible to all researchers.</p> / Master of Science (MSc)

Le cycle cellulaire est l'ensemble des étapes qui conduisent une cellule mère à se diviser en deux cellules filles. La protéine Checkpoint kinase 1 (Chk1) est importante pour sa progression. Nous avons d'une part cherché à savoir si Chk1 intervenait lors des mécanismes de production des plaquettes, car ces cellules permettant la coagulation du sang sont issues d'un cycle cellulaire particulier. Par ailleurs, nous avons étudié le rôle de Chk1 dans la Leucémie Aiguë Myéloïde (LAM), cancer des cellules sanguines. Les patients atteints de LAM sont traités par une chimiothérapie visant à endommager l'ADN afin d'entrainer la mort des cellules cancéreuses. Chk1 est garante du contrôle de la réparation des dommages de l'ADN, ce qui contrecarre l'effet de la chimiothérapie. Elle pourrait donc favoriser l'apparition de résistance. Son rôle dans les LAM étant peu connu, l'objectif de ce projet est donc de vérifier si Chk1 favorise la résistance des cellules leucémiques aux chimiothérapies. / The cell cycle is a series of events that takes place in a mother cell, leading to its division into two daughter cells. The protein Checkpoint kinase 1 (Chk1) is mandatory for its coordinated progression. In this PhD projet, we wondered on the one hand whether Chk1 could be involved in the platelets production process, because these componants of blood that enables coagulation are produced due to a particular cell cycle dedicated to this end. On the other hand, we studied the role of Chk1 in Acute Myeloid Leukemia (LAM) physiopathology. LAM is a cancer of blood cells, in which patients are treated with drugs that create DNA damages, causing the death of tumoral cells. The role of Chk1 in the drug response in LAM is not well studied, but, as it enables DNA repair, it may render theses medicines less efficient, leading to relapses to therapies. So the goal of this project is to check wether Chk1 favors the resistance of some LAM cells to chemotherapeutic treatments.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13159号 / 論医博第2146号 / 新制||医||1029(附属図書館) / (主査)教授 河本 宏, 教授 前川 平, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

La greffe de cellules souches hématopoïétiques est parfois le seul traitement efficace contre les cancers hématologiques ainsi que plusieurs autres désordres reliés au système hématopoïétique. La greffe autologue est souvent le traitement de choix pour les patients atteints de lymphome ou de myélome. Dans ce cas, les cellules souches hématopoïétiques (CSH) du patient sont récoltées et congelées. Le patient subit ensuite des traitements de chimiothérapie et/ou radiothérapie qui éliminent les cellules malignes, mais détruisent aussi son système hématopoïétique. Ce dernier sera ensuite reconstitué par la greffe de CSH. Ces traitements ont pour conséquence de plonger le patient en état d’aplasie pour une période variant de 2 à 4 semaines. La thrombocytopénie (faible taux de plaquettes) est une complication majeure nécessitant des transfusions plaquettaires répétées et associée à une augmentation de la mortalité hémorragique post-transplantation. Il serait particulièrement intéressant de développer une thérapie accélérant la reconstitution des mégacaryocytes (MK), ce qui aurait pour effet de raccourcir la période de thrombopénie et donc de diminuer les besoins transfusionnels en plaquettes et potentiellement augmenter la survie. HOXB4 est un facteur de transcription qui a déjà démontré sa capacité à expandre les CSH et les progéniteurs multipotents (CFU-GEMM) donnant naissance aux MK. Il est donc un bon candidat pour l’expansion des progéniteurs MK. Comme la protéine HoxB4 a par contre une courte demi-vie (~1.1h), des protéines HoxB4 de deuxième génération avec une plus grande stabilité intracellulaire ont été créées (1423 (HoxB4L7A), 1426 (HoxB4Y23A) et 1427 (HoxB4Y28A)). Nous avons donc étudié la capacité d’HoxB4 sauvage et de deuxième génération à expandre les CSH, ainsi que les MK donnant naissance aux plaquettes. La surexpression rétrovirale de ces protéines HoxB4Y23A et HoxB4Y28A conduit à une expansion des progéniteurs MK murins in vitro supérieure à HoxB4-wt, 1423 et au contrôle GFP. La reconstitution plaquettaire in vivo dans un modèle murin a ensuite été évaluée par des transplantations primaires et secondaires. Les résultats révèlent que la surexpression rétrovirale des différents HoxB4 n’apporte pas de bénéfice significatif à la reconstitution plaquettaire des souris. Lorsque cultivées dans un milieu favorisant la différenciation mégacaryocytaire, le traitement de cellules CD34+ dérivées du sang de cordon ombilical avec les protéines recombinantes TATHoxB4WT ou de seconde génération n’a pas augmenté la production plaquettaire. Par contre, de manière intéressante, les cellules CD34+ provenant de sang mobilisé de patients atteints de myélome et mises en culture dans un milieu favorisant l’expansion des CSH ont montré des différences significatives dans la différenciation des progéniteurs MK en présence de la protéine recombinante TATHoxB4. La protéine HOXB4 possède donc un avenir prometteur quant à une amélioration de l’état thrombocytopénique chez les patients. / Haematopoietic stem cell (HSC) transplantation is the most efficient treatment against a number of cancers or other disorders of the hematologic system. Prior to HSC transplantation, patients are exposed to high doses of radiotherapy and/or chemotherapy to eliminate malignant cells. However, these treatments result in a state of aplasia, particularly in thrombocytopenia, which is characterised by very low blood platelet counts. Platelets produced by megakaryocytes (MK) are essential components of the blood system and play a critical role in the prevention of bleeding. Thus a low platelet blood level is a major complication and contributes significantly to transplant related mortality. At present, regular infusion of platelets isolated from healthy donors is the treatment of choice for thrombocytopenia. However, this is cumbersome for patients as well as donors and, in many instances results in platelet refractoriness due to the generation of auto-antibodies against disparate HLA molecules expressed on donor platelets. Therefore, the development of strategies to accelerate MK production and thus platelet reconstitution post HSC transplant would represent a major advance. It has already been shown that HoxB4 expands HSC and multipotent progenitors (CFU-GEMM) that give rise to megakaryocytes (MK). Thus HoxB4 is a great candidate for in vitro MK progenitor expansion. However, the short half-life of HoxB4 protein prompted us to generate a second generation of HoxB4 proteins with greater intracellular stability. We therefore studied the capacity of wild type (WT) and HoxB4 with 3 substitutions (1423 (HoxB4L7A), 1426 (HoxB4Y23A) and 1427 (HoxB4Y28A) resulting in a longer protein half-life to expand HSC as well as MK progenitors. Retroviral-mediated expression of HoxB4Y23A and HoxB4Y28A proteins showed a greater expansion of murine MK progenitors, in comparison with HoxB4WT or HoxB4L7A proteins or GFP control. We also evaluated the ability of HSC expressing second generation HoxB4 to generate platelets in a murine model. Our results show that retroviral-mediated transduction of second generation HoxB4 in murine HSC does not provide a significant advantage over HoxB4WT in platelet reconstitution in mice. Interestingly, treatment of CD34+ cells derived from cord blood showed only marginal effect of HoxB4WT or second generation HoxB4 soluble recombinant proteins when cultured under conditions optimized for megakaryocyte differentiation. Unexpectedly, CD34+ cells derived from mobilized peripheral blood of myeloma patients showed a significant increase in MK progenitor differentiation in the presence of TAT-HoxB4WT when cultured in expansion medium for HSC. Thus, HoxB4 holds promise in autologous HSC transplantation for the treatment of thrombocytopenic patients.