No description available.

Clonal haematopoiesis of indeterminate potential, or CHIP are a family of mutations present in the general population. CHIP-mutations are prevalent in the haematopoietic stem cells and in the more mature cell populations, T-lymphocytes, B-lymphocytes and myeloid cells (CD3+, CD19+ and CD33+ cells) in blood. By separating these cell populations using magnetic isolation, extracting DNA from the cell populations, and detecting the same mutation in all cell populations, one can prove the presence of CHIP-mutations in a hematopoietic stem cell. At least 50 ng good quality DNA is needed for the gene analysis to detect CHIP-mutations. The magnet separated cell population may be very small, so the DNA extraction method must be optimized to achieve enough DNA yield. The main purpose of the method development was to compare two storage methods before DNA-extractions, and then three different DNA-quantification methods after the DNA-extractions. After the best storage and quantification methods were identified, five samples of cryo-preserved viable cells were used to isolate cell populations using magnetic beads covered in specific antibodies and a magnetic field, and then quantified. Results of the study showed that the best way was to store the cells in ATL-buffer and Proteinase K. To quantify DNA, qPCR was the most accurate method, since the other methods showed incorrect results because of the low DNA concentrations. Magnet cell separation was partly successful. All except one of the DNA yields from the cell separation protocols reached the critical amount of DNA, but some yields were not pure yields of the sought-after cell population. In general, the method must be worked on more with further research.

Graft-versus-host disease (GvHD) is a frequent, and often life-threatening, complication after an allogeneic, hematopoietic stem cell transplantation (allo-SCT). It can appear in an acute or a chronic form and presents different grades of severity. Particularly, the severe forms of GvHD are often responsible for a change of the curative intent for allo-SCT into a palliative goal of care. For this non-systematic review, we conducted a focused literature search in the MEDLINE database via PubMed to examine whether patients with severe forms of GvHD might have special needs and burdens from a supportive and palliative care perspective. To draw a comprehensive picture of this patient group, we included findings on quality of life (QoL) and physical symptoms and function as well as psychological and spiritual well-being. In most domains, patients with severe forms of GvHD showed greater impairment and a higher symptom burden compared to patients with milder forms of GvHD. However, we could not identify any studies that specifically investigated patients with severe forms of GvHD. Further research in this field is necessary to guarantee the highest standard of care for this very special patient group.

No description available.

Hematopoietic stem cells (HSCs) are critical to the development of the hematopoietic system during ontogeny and maintaining hematopoiesis under steady-state. Several genes implicated in asymmetric cell division (ACD) have been found to influence HSC self-renewal in normal hematopoiesis and various leukemias. From a separate survey of genes associated with ACD, I now present the results from dedicated functional studies on two genes – Arhgef2 and Staufen1 – in HSCs and identify their potential contributions to benign hematopoietic disorders. Specifically, I present evidence that demonstrates a conserved role of Arhgef2 in orienting HSC division, the loss of which leads to HSC exhaustion that may underlie and contribute to the pathogenesis of Shwachman-Diamond syndrome. I also identify Staufen1 as a critical RNA-binding protein (RBP) in HSC function, downregulation of which elicits expression signatures consistent with clinical anemias reminiscent of aplastic anemia and/or paroxysmal nocturnal hemoglobinuria. I end by reviewing how RBPs function in HSCs and discuss future research directions that could further elucidate how bone marrow failure syndromes arise at the stem cell level. / Thesis / Doctor of Philosophy (PhD)

Background: Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell–cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication. Results: Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34+ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34+ progenitors, leukemic cells can take up migrasomes. Conclusion: Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment.

DNS-Methylierung ist ein zentraler epigenetischer Prozess, der essentiell für die Differenzierung embryonaler Stammzellen ist, über dessen Funktion in somatischen Zellen allerdings wenig bekannt ist. In der vorliegenden Doktorarbeit wurden zwei Mausmodelle analysiert, um die Rolle der durch DNS Methyltransferase 1 (DNMT1) hergestellten DNS-Methylierung im adulten hämatopoetischen System zu untersuchen. Als erstes wurde ein „Knockout“-Modell gewählt, um DNMT1 im hämatopoetischen System zu eliminieren. Des Weiteren wurde eine Mausmutante mit reduzierter DNMT1 Expression analysiert. Die vollständige Entfernung von DNMT1 aus dem hämatopoetischen System adulter Mäuse resultierte in Zytopenie und Anämie, gefolgt vom raschen Tod aller Tiere. Die Analyse des Knochenmarks dieser Mäuse zeigte einen fast vollständigen Verlust von hämatopoetischen Stamm- sowie Vorläuferzellen. Dies zeigt, dass die durch DNMT1 erzeugte DNS-Methylierung essentiell für Homöostase und Differenzierung von hämatopoetischen Stammzellen ist. Mäuse mit reduzierter DNMT1 Expression hingegen sind lebensfähig und zeigen einen niedrigen Grad an DNS-Methylierung in verschiedensten Geweben, einschließlich des hämatopoetischen Systems. Durch eine detaillierte phänotypische und funktionelle Analyse der hämatopoetischen Stammzellen zeigte sich, dass der veränderte DNS-Methylierungsgrad ein vermindertes Selbsterneuerungspotenzial zur Folge hat. Interessanterweise fehlen DNMT1 hypomorphen Mäusen lymphoide Vorläuferzellen sowie reife lymphoide Zellen, wohingegen myeloide und erythroide Zellpopulationen keine Veränderungen zeigten. Genomweite Expressionsanalysen von Stammzellen sowie myeloiden Vorläuferzellen zeigten, dass hypomethylierte Stammzellen eine verfrühte myeloerythroide Entwicklung vollziehen und liefern damit eine Erklärung für den Verlust des Selbsterneurungspotenzials und der lymphoiden Entwicklung. Diese Resultate identifizieren eine bis hierhin unbekannte Funktion von spezifischen DNS-Methylierungsgraden für die Steuerung von funktionellen Programmen wie Selbsterneuerung und Differenzierung in hämatopoetischen Stammzellen. / DNA methylation is one of the major epigenetic mechanisms which is known to play a role in embryonic stem cell fate, but its function in somatic stem cells is not well understood. In this thesis two different genetic mouse models were chosen to address the role of DNA methyltransferase 1 (DNMT1) controlled DNA methylation in adult hematopoiesis. First, a conditional knockout approach was used to delete DNMT1 in the adult hematopoietic system. Second, DNMT1 hypomorphic mice with reduced DNMT1 expression were analyzed. Complete DNMT1 deletion in hematopoietic cells led to severe cytopenia and anemia causing rapid lethality of all animals. Bone marrow analysis revealed an almost complete absence of hematopoietic stem and progenitor cells in DNMT1 ablated primary mice as well as in secondary chimeric mice. These results indicated that DNMT1 controlled maintenance of DNA methylation is indispensable for HSCs preservation and differentiation. In contrast to complete DNMT1 deletion, mice with hypomorphic DNMT1 expression were viable, but showed low methylation levels in multiple tissues including the hematopoietic system. Detailed phenotypical and functional analysis of the hypomethylated hematopoietic stem cell (HSCs) compartment revealed an impaired homeostasis and self-renewal capacity. Intriguingly, mutant animals had profoundly reduced lymphoid cell compartments, whereas myeloid and erythroid compartments were unchanged. Expression profiling of stem and myeloid progenitor cells unexpectedly demonstrated that reduced DNA methylation forces the HSC to adopt a myeloid lineage identity. These results, showing the inability of hypomethylated HSCs to maintain an undifferentiated state, provided an explanation for their disturbed capability to self-renew and produce lymphocytes. Taken together, these findings suggest that distinct levels of DNA methylation are required to control different functional programs such as self-renewal and alternative lineage choices in HSCs, thus uncovering a previously unrecognized function for DNMT1 activity.

Dogs are useful preclinical models for the translation of cell transplantation therapies from the bench to the bedside. In order for canine models to be utilized for stem cell transplantation research, it is necessary to advance discoveries in the fields of canine stem cell biology and transplantation. The use of side population hematopoietic stem cells (HSCs) has garnered much interest for the purification of mouse HSCs and has been translated to several other species, including human. In order to assess if this method of purification of HSCs could be useful for stem cell therapies in humans, safety and efficacy studies in a large animal model, such as the dog would be required. With this objective in mind, we isolated canine bone marrow-derived side population (SP) stem cells and assessed their multilineage differentiation in vitro and engraftment potential in vivo. Utilizing a pregating strategy to enrich for small, agranular SP cells we were able to enrich for blast cells, expressing the ABCG2 transmembrane pump known to be associated with murine and human SP cells. Canine SP cells were also enriched for C-KIT positive cells and lacked expression of CD34 as identified in other species. The small, agranular SP fraction had high CFU potential after long-term culture with canine bone marrow stromal cells and cytokine supplementation. Yet, canine SP cells demonstrated low-level engraftment within the NOD/SCID-β2m-/- xenotransplantation model as compared to unfractionated canine bone marrow, which was indicative of suboptimal activation of quiescent canine SP cells within the murine bone marrow niche. A second source of transplantable canine stem cells was examined through the derivation of canine embryonic stem cells (cESCs). The cESC lines described herein were determined to have similar pluripotent stem cell characteristics to human embryonic stem cells, in that they were maintained in an undifferentiated state upon extended passaging as determined by their expression of the human stem cell markers, OCT3/4, NANOG, SOX2, SSEA3, SSEA4, TRA1-60, TRA1-81 and alkaline phosphatase. In addition, cESCs could be induced to differentiate to cells of the three germ layers within in vitro embryoid body cultures and adherent differentiation cultures. Importantly, these cESC lines were the first reported to differentiate in vivo within teratomas. One method of transplanting stem cells to canine recipients involves the delivery of donor cells to the yolk sacs of developing fetuses in utero. Utilizing cells labeled with supraparamagnetic particles conjugated to a Dragon Green fluorophore and the intracellular fluorescent dye, CMTMR, donor cells were tracked from the yolk sac injection site to fetal tissues after transplantation in early (day-25) and mid (day-35) gestation canine fetuses. Labeled cells were localized primarily to the fetal liver and developing bone marrow cavities when examined at gestational day 32, and had been redistributed to not only the fetal liver and bone marrow by day 42, but also to nonhematopoietic tissues, including the lungs and hearts. No labeled cells were detected within the yolk sacs of transplanted fetuses at either time point. These studies demonstrated the efficacy of yolk sac in utero transplantation for the delivery of donor cells to fetal tissues. Collectively, these results indicate that canine stem cells with characteristics similar to human can be isolated and their engraftment, proliferation and differentiation may be assessed in future studies utilizing the canine in utero transplantation model employing yolk sac delivery.

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.

La greffe de sang de cordon est de plus en plus utilisée et a permis de traiter avec succès chez l’enfant des déficits immunitaires ainsi que des hémopathies malignes comme les leucémies. Malgré d’importants avantages tels que l’absence de risque pour le donneur ou la plus faible incidence de maladie du greffon contre l’hôte (GvHD), utiliser le sang de cordon comporte certains inconvénients. En effet, une reconstitution immunitaire retardée, des infections opportunistes en plus grand nombre et un risque de rechute sont des complications qui peuvent survenir et engendrer un risque pour le pronostic vital du patient. Par conséquent, de nouvelles stratégies d’immunothérapies doivent être envisagées. Dans le cadre de ce travail, nous nous sommes particulièrement intéressés aux cellules dendritiques plasmacytoides (pDC) dont les fonctions sont importantes pour l’initiation des réponses immunitaires innée et adaptative et particulièrement pour leur capacité à activer les cellules NK. Afin d’élucider le rôle et l’impact de ces cellules dans les greffes de sang de cordon, le nombre et la fonction des pDC et des NK a été suivi longitudinalement chez des patients ayant subi une greffe de sang de cordon comparativement à des patients transplantés avec de la moelle osseuse. Nous avons ainsi démontré que les pDC et les NK apparaissent précocement suite à une greffe de sang de cordon et que ces cellules sont fonctionnelles. Ces résultats mettent donc en lumière que ces cellules pourraient être de bons outils pour l’établissement d’une immunothérapie après greffe de sang de cordon. De plus, la caractérisation fonctionnelle des pDC du greffon de sang de cordon a permis de révéler une plus faible production d’IFN-α par les pDC, comparativement aux pDC de sang d’adulte. Cette différence pourrait jouer un rôle dans la plus faible incidence de GvHD après les greffes de sang de cordon. Dans le but de préciser les mécanismes moléculaires de régulation négative de la production d’IFN-α par les pDC de sang de cordon, nous avons étudié les protéines de la voie de signalisation TLR9-IRF7. L’expression similaire de l’ARN du TLR9, MyD88, IRAK1 et IRF7 contraste avec la plus faible expression des protéines correspondantes. De plus, l’expression des MicroARNs miR-146a et miR-155 est plus élevé dans les pDC de sang de cordon comparativement aux pDC de sang d’adultes. Ensemble, ces données pointent une régulation négative post-transcriptionnelle de la voie TLR9-IRF7 qui pourrait expliquer la plus faible production d’IFN-α des pDC du sang de cordon. L’ensemble des ces travaux suggère que les pDC pourraient représenter une cible de choix dans le développement de nouvelles approches thérapeutiques dans les greffes de sang de cordon. / Umbilical cord blood transplantation has increasingly been used as a source of hematopoietic stem cells to successfully treat immunodeficiencies and malignant diseases such as leukemia in pediatric patients. Despite important advantages, namely lack of risk for the donor and low incidence of GvHD, use of cord blood is associated with several drawbacks. Specifically, delayed immune reconstitution, more opportunistic infections and a relative risk of relapse are complications that may occur and lead to a poor prognosis. Consequently, new immunotherapeutic strategies should be considered. In this study, we were interested in plasmacytoid dendritic cells (pDC), whose functions are important for initiation of innate and adaptive immune responses and, in particular, for their ability to activate natural killer cells (NK). In order to elucidate the role and the impact of these cells in cord blood transplantation, pDC and NK numbers and function have been longitudinally followed in cord blood and bone marrow recipients. We showed that pDC and NK cells appeared early after umbilical cord blood transplantation and that these cells retained functional activity. Thus, these cells may constitute a good tool for immunotherapy in umbilical cord blood transplantation. Moreover, the functional characterization of pDC in cord blood revealed a lower production of IFN-α by cord blood pDC, which may play a role in the lower incidence of GvHD after umbilical cord blood transplantations. In order to determine the molecular mechanism for the negative regulation of IFN-α production by cord blood pDC, we studied the expression of TLR9-IRF7 pathway. The stable expression of TLR9, MyD88, IRAK1 and IRF7 mRNA contrasts with the lower expression of corresponding proteins. Interestingly, expression of microRNA miR-146a and miR-155 is higher in cord blood pDC. Together, these results point to a post-transcriptionnal negative regulation of TLR9-IRF7 pathway which may explain the lower IFN-α production by cord blood pDC. This work reinforces the idea that pDCs constitute a target of choice for developing new therapeutic approaches in cord blood transplantations.