Master of Science / Haematopoietic stem cell transplantation (HSCT) is a complicated and high-risk procedure used to cure disease or stop the spread of disease in a range of cancers. HSCT carries a high incidence of mortality and is associated with distressing short and long-term side effects. In addition, patients remain at risk of recurrence or mortality years after transplantation. Therefore, patients undergoing HSCT have been found to experience significant emotional and psychosocial distress because of the trauma associated with treatment. The literature suggests that about 50% of HSCT patients will experience clinical levels of distress. Carers and family members play an important role in caring for these ill patients in the short and long-term. Major role changes and financial stressors are experienced in many families, adding to the burden of care. However, very little is known about the rates of psychopathology in carers of HSCT patients. Due to the arduous nature of HSCT, psycho-educational programmes have been developed by major transplant centres and hospitals in order to provide HSCT patients and their families with information on the treatment process, side effects, risks, and long-term outcomes. Research on patient education in oncology has shown that providing patients and carers with information about their illness and treatment reduces anxiety and distress. To date, there have been no empirical evaluations to support the use of education programmes for HSCT patients. While it could be assumed that information would be helpful in reducing anxiety and depression in HSCT as it is in oncology generally, the information provided to these patients is usually more confronting and therefore, may be less reassuring. Thus, it is not known whether providing patients with education about HSCT reduces patient and carer distress or whether it might actually increase adverse outcomes. The aim of the present study was to evaluate the rates and correlates of distress in carers and patients and examine the effect of a psychoeducation programme for patients undergoing HSCT and their carers on knowledge, distress, information satisfaction, social support and caregiver burden. A randomised control trial was conducted to provide empirical data in relation to the latter aim. The following hypotheses were proposed. Firstly, it was hypothesised that patients and carers who received the education programme would have higher levels of knowledge, not evidenced in a group waiting to receive the programme. Secondly, it was hypothesised that the education program would not lead to increased anxiety and depressive symptoms. Thirdly, patients who know more about their condition would be the least distressed. As predicted, this study found high levels of distress, particularly in carers. Higher patient distress was related to having more concern about one’s illness and experiencing more symptoms. Education was effective in increasing patient and carer knowledge. Importantly there were no adverse effects of knowledge and greater patient knowledge following the education program was associated with less distress, although there was no direct effect of education on distress. Future research should aim to identify what aspects of the education program are helpful to patients. Finally, support interventions such as CBT are needed to help patients and carers, in particular, cope with the high levels of distress inherent in the HSCT experience.

The regenerative potential of adult stem cell populations within the human body bears great promises for their use in regenerative medicine. The bone marrow (BM) harbours two different types of adult stem cells, haematopoietic stem and progneitor cells (HSPCs) and multipotent mesenchymal stromal cells (MSCs), which are tightly regulated in their distinct anatomically defined niches by multiple cues such as cytokines, cell-cell contacts, the extracellular matrix (ECM) and the physical microenvironment. The ex vivo expansion of these cells for applications in regenerative therapies is of great interest and several biomaterial approaches attempt to mimic the natural BM niche and its components to control stem cell maintenance and differentiation. However, as of now the complexity of such stem cell niches is hard to recapitulate. Towards this goal, this work was focussing on the ECM environment of BM stem cells and was set out to engineer improved in vitro culture systems. MSC themselves are one of the most important cell types within the BM that secrete and construct ECM-networks and thereby shape the microenvironment of the residing cells. The potential of primary human BM-MSC to secrete ECM in vitro has been exploited to generate niche-like ECM surrogates in a robust and versatile format. Application of decellularisation regimes allowed the fabrication of complex matrices which demonstrated suprastructural, compositional and physicochemical properties compareable to those of the native BM-ECM environment. Reliable stability and reproduciblity was achieved by a dedicated procedure of maleic anhydride co-polymer-mediated covalent binding of fibronectin and subsequent anchorage of cell-secreted ECM molecules. As a result of the high reproducibility, a complete proteomic register of ECM molecules was obtained in combination with determining the complex fibrillar and soft gel-like characteristics of MSC-derived matrices. Based on the established BM niche-like substrate, the impact of extracellular matrices on MSC and HSPC ex vivo behavior has been explored. Both cell types demonstrated strong adhesion to ECM substrates and depicted a changed cellular morphology upon contact with native ECM structures compared to standard culture substrates or simple ECM protein coatings, indicating an intense interplay between the cell and the microenvironment. MSC that re-grew into their own matrices have shown advantageous proliferation and cytokine secretion levels as well as enhanced differentiation intensity (upon differentiation induction) compared to MSC that were cultured on less complex substrates. Similarly, HSPC were also instructed for enhanced expansion on MSC-derived matrices without exhaustion of stem cell-marker expressing progenitor cells. The efficiency of these matrices was related to their ability to mimic the native composite suprastructure, ligand nano-topography, molecular composition and physical properties of natural BM ECM environments. The data obtained within this thesis set the ground for a more rational design of artificial stem cell niches with defined and distinct properties, offering exciting options for the in-depth analysis and understanding of stem cell regulation by exogenous cues.

Acute graft versus host disease (aGVHD) affects more than 40% of patients undergoing haematopoietic stem cell transplantation. aGVHD occurs after transplantation of donor haematopoietic cells into hosts incapable of rejecting the donor cells, when donor T cells attack host tissue. Despite extensive efforts, aGVHD remains problematic to prevent and difficult to control. Current therapies to prevent aGVHD induce profound immunosuppression, leaving patients at increased risk of infection and leukaemic relapse. Dendritic cells (DC) are professional antigen presenting cells of haematopoietic origin and are the primary stimulators of the immune system, uniquely being able to activate naïve T cells. A growing body of evidence suggests that DC are responsible for the stimulation of the donor T cells which cause aGVHD. I have used a model of aGVHD which utilizes conditioned severe combined immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMC). In this model human CD4+ T cells appear to be responsible for an aGVHD-like syndrome which results in death 15-30 days post transplant. I have shown, using in vitro depletion of individual populations, that other subpopulations of human PBMC did not affect the survival of the mice. I have also demonstrated that human DC are required for the induction of aGVHD in the majority of mice. This novel finding validated the use of this model to test the primary hypothesis; that antibody mediated depletion of DC would prevent aGVHD. The murine IgM monoclonal antibody (Mab), CMRF-44 Mab, is specific for an unknown molecule expressed on the surface of activated human DC. Previous work had shown that when mixed lymphocyte reaction stimulator cells were depleted of CMRF-44+ cells, there was a significant reduction in the proliferation of responder cells. Here I tested the efficacy of CMRF-44 as a therapy for the prevention of aGVHD in the model. CMRF-44 Mab did not improve survival of mice treated with human PBMC, despite recent data showing that CMRF-44 expression on DC was predictive of aGVHD in patients. In vitro depletion of CMRF-44+ cells from human PBMC prior to transplantation also did not reduce incidence of aGVHD. An alternate target for the depletion of human DC was CD83 which is also expressed on the surface of activated human DC. I generated a rabbit polyclonal antibody using a human CD83 fusion protein, which was then affinity purified in a multi-step process which yielded only antibody specific for human CD83. Treatment with this antibody greatly improved survival of transplanted mice. Further experiments showed that anti-CD83 treatment did not abrogate human leucocytes including CD8+ memory T cells suggesting that a therapy using an anti-CD83 antibody has the potential to prevent aGVHD without the immunosuppression associated with current anti-aGVHD therapies. The work described here has validated the use of a human mouse chimeric model as an in vivo assay of human DC function and shown that targeting CD83 has the potential to reduce the incidence of clinical aGVHD whilst preserving donor memory T cells.

Master of Science / Haematopoietic stem cell transplantation (HSCT) is a complicated and high-risk procedure used to cure disease or stop the spread of disease in a range of cancers. HSCT carries a high incidence of mortality and is associated with distressing short and long-term side effects. In addition, patients remain at risk of recurrence or mortality years after transplantation. Therefore, patients undergoing HSCT have been found to experience significant emotional and psychosocial distress because of the trauma associated with treatment. The literature suggests that about 50% of HSCT patients will experience clinical levels of distress. Carers and family members play an important role in caring for these ill patients in the short and long-term. Major role changes and financial stressors are experienced in many families, adding to the burden of care. However, very little is known about the rates of psychopathology in carers of HSCT patients. Due to the arduous nature of HSCT, psycho-educational programmes have been developed by major transplant centres and hospitals in order to provide HSCT patients and their families with information on the treatment process, side effects, risks, and long-term outcomes. Research on patient education in oncology has shown that providing patients and carers with information about their illness and treatment reduces anxiety and distress. To date, there have been no empirical evaluations to support the use of education programmes for HSCT patients. While it could be assumed that information would be helpful in reducing anxiety and depression in HSCT as it is in oncology generally, the information provided to these patients is usually more confronting and therefore, may be less reassuring. Thus, it is not known whether providing patients with education about HSCT reduces patient and carer distress or whether it might actually increase adverse outcomes. The aim of the present study was to evaluate the rates and correlates of distress in carers and patients and examine the effect of a psychoeducation programme for patients undergoing HSCT and their carers on knowledge, distress, information satisfaction, social support and caregiver burden. A randomised control trial was conducted to provide empirical data in relation to the latter aim. The following hypotheses were proposed. Firstly, it was hypothesised that patients and carers who received the education programme would have higher levels of knowledge, not evidenced in a group waiting to receive the programme. Secondly, it was hypothesised that the education program would not lead to increased anxiety and depressive symptoms. Thirdly, patients who know more about their condition would be the least distressed. As predicted, this study found high levels of distress, particularly in carers. Higher patient distress was related to having more concern about one’s illness and experiencing more symptoms. Education was effective in increasing patient and carer knowledge. Importantly there were no adverse effects of knowledge and greater patient knowledge following the education program was associated with less distress, although there was no direct effect of education on distress. Future research should aim to identify what aspects of the education program are helpful to patients. Finally, support interventions such as CBT are needed to help patients and carers, in particular, cope with the high levels of distress inherent in the HSCT experience.

Embryonic haematopoiesis is a complex process under intensive research. Murine definitive Haematopoietic Stem Cells (HSCs) originates from the Aorta-Gonad-Mesonephros (AGM) region of E10.5 embryo. It is thought that definitive HSCs arise from endothelial lining of dorsal aorta. However, detail of HSC specification in the developing embryo remains elusive. One way to deciphering events occurred during HSC specification is to derive cell lines from the developing HSC niche. Previous work by Oostendorp et al. showed the AGM and fetal liver derived lines could maintain HSCs in vitro (Oostendorp, Harvey et al. 2002). In this study, I established a more robust immortalization system using normal SV40 large T antigen delivered via Neon™ electroporation system. The new immortalization system achieved direct immortalization without going through crisis. And it is compatible with small number of primary cells dissected from different haematopoietic niches. With my new system, multiple cell lines from different haematopoietic sites at different developmental points are derived. Moreover, some of these lines demonstrated ability to mature precursors from E9.5 embryo (pro-HSCs) to definitive HSC without help of growth factors. This result is better compared to OP9 stromal lines. Such data proved usefulness of using stromal cell lines to study haematopoietic specification.

No description available.

L’hématopoïèse est le processus de formation des cellules souches hématopoïétiques (CSH); elle est conservée au cours d’évolution. Durant l’hématopoïèse embryonnaire, deux vagues hématopoïétiques se succèdent, la vague primitive et la vague définitive. La vague primitive produit des macrophages, des neutrophiles et des érythrocytes. Au cours de la vague définitive, les CSH émergent du plancher de l’aorte dorsale par une transition endothélio-hématopoïétique ou TEH, dans une région appelée aorte-gonades-mesonephros (AGM).Ces dernières années, des études des organes hématopoïétiques chez les mammifères ont démontré que le microenvironnement joue un rôle crucial dans l’émergence et le devenir des CSH. Pendant ma thèse, je me suis intéressée au rôle du microenvironnement dans la mise en place de l’hématopoïèse définitive chez l’embryon de zebrafish. Dans l’AGM, j’ai caractérisé et évalué la contribution de différents acteurs dont deux populations en particulier, les macrophages et le système neuronal sympathique. Chacune de ces cellules joue un rôle spécifique durant la vague définitive de l’hématopoïèse. Les macrophages mobilisent des CSH de l’AGM afin de permettre leur intravasation et la colonisation des organes hématopoïétiques. Les catécholamines synthétisées par le système neuronal sympathique quant à elles contrôlent la TEH par l’activation des récepteurs beta2b et beta3 dans l’AGM.En conclusion, nous avons démontré que le microenvironnement influence l’hématopoïèse définitive chez le zebrafish par différents mécanismes. Ces travaux ont pour objectif d’améliorer la compréhension du mécanisme de genèse des CSH et potentiellement de permettre un jour la production de CSH in vitro. / Haematopoiesis is the process of haematopoietic stem cell (HSC) generation conserved in all vertebrates. During the embryonic development, two successive waves of haematopoiesis occur – the primitive and the definitive wave. The first one gives rise to erythrocytes, macrophages and neutrophils. During the second one, HSCs emerge from the ventral wall of dorsal aorta (DA) in the aorta-gonads-mesonephros (AGM) region by a process called endothelial-to-haematopoietic transition or EHT.In the last years, several studies performed in mammals have shown that the microenvironment plays a key role in haematopoiesis. During my thesis I have studied the role of the microenvironment in definitive haematopoiesis in the zebrafish embryo. I have described several cell components present in the AGM and evaluated their contribution to the haematopoiesis. I further analysed two of those players: macrophages and sympathetic nervous system. Each of them plays a specific role during the definitive wave of haematopoiesis. Macrophages mobilise nascent HSCs from the AGM to allow their intravasation and colonisation of haematopoietic organs. Catecholamines synthetized by sympathetic nervous system control EHT through the activation of beta2b and beta3 receptors in the AGM.In conclusion, we have shown that the microenvironment can substantially influence the definitive haematopoiesis in the zebrafish by distinct mechanisms. These findings would help to understand the mechanism of HSC generation and potentially to allow in vitro HSC production.

The Mucopolysaccharide (MPS) diseases are a group of lysosomal storage disorders, caused by a lack of the enzymes required for catabolism of glycosaminoglycans (GAGs), leading to severe neurological decline, skeletal deformities, organomegaly, cardiac and respiratory compromise, and premature death. The severe form of MPS I, Hurler syndrome, can be successfully treated using haematopoietic stem cell transplantation (HSCT), but the risks associated with myeloablation and immune suppression limit the broader application of HSCT to attenuated diseases. Successful engraftment in MPS I has been difficult to achieve, and requires fully myeloablative conditioning, whilst reduced intensity conditioning is a risk factor for graft rejection. Non-myeloablative conditioning generating reliable graft acceptance and high donor chimerism could increase safety and applicability of HSCT in genetic disease, therefore the aim of this research was to identify such a regimen in a clinically relevant mouse model of HSCT.Conditioning regimens developed in existing mouse models of HSCT have had limited clinical success, and often require clinically unachievable high cell doses or less stringent strain combinations to overcome allogeneic transplant rejection. To improve clinical relevance we used CBA donors and C57BL/6 recipients, which require full myeloablation with busulfan and immune suppression using non-depleting anti-CD4 and anti-CD8 monoclonal antibodies for engraftment of low cell doses across a major histocompatibility complex barrier. In syngeneic transplant donor chimerism was improved by generating a greater ratio of donor:recipient haematopoietic cells in the bone marrow initially, therefore we tested granulocyte colony stimulating factor (G-CSF), high cell dose and stem cell niche disruption and compared this to anti-CD40L costimulatory blockade in allogeneic transplant performed with a reduced dose of busulfan that was insufficient for graft acceptance. Despite improvements in initial engraftment with some of these treatments, only combined signal 1 and 2 T cell blockade were effective in reducing the dose of busulfan required for long-term graft acceptance. Early detection of MPS is important in treatment success; good disease biomarkers are vital, and biomarkers suitable for monitoring treatment outcome in MPS are lacking. We evaluated serum heparin cofactor II-thrombin (HCII-T) complex for MPS. We determined optimal sample collection and storage conditions, assay limitations and developed measurement in dried blood spots. Dermatan sulphate has a greater effect on in vivo HCII-T complex formation than heparan sulphate, thus in the MPS mouse models HCII-T is a reliable biomarker for MPS I, but not MPS IIIA or IIIB. HCII-T is greatly elevated in MPS I, II and VI patients, who all store dermatan sulphate, but it is also elevated by a small but significant amount in MPS III patients, who store heparan sulphate. HCII-T was also measured longitudinally in MPS I, II and VI patients, compared to an existing clinical biomarker, and validated against clinical outcomes to show that it is a good biomarker of short-term treatment outcomes and responds rapidly to perturbations in treatment. Finally, we determined whether an engraftment defect was observed in the MPS I mouse model, and show that this is present following both syngeneic and allogeneic HSCT. The effect of enzyme replacement therapy (ERT) and anti-inflammatory treatment prior to allogeneic HSCT was investigated, and initial results suggest that ERT, but not ibuprofen, may improve HSCT outcome. Overall, a clinically relevant mouse model of allogeneic HSCT has been developed and used to determine a non-myeloablative conditioning regimen that generates high levels of donor chimerism with a minimal dose of busulfan and blockade of both signal 1 and 2 of T cell activation. The conditions required to observe an engraftment defect in MPS I mice have also been defined, and preliminary studies have suggested that ERT, but not anti-inflammatory treatment, may overcome the engraftment defect in MPS I. Alongside this work, the HCII-T biomarker has been evaluated in MPS mouse models and patients, determining that it correlates well with short-term treatment outcomes. The techniques and models developed here will provide an excellent basis for further work in developing non-myeloablative conditioning for bone marrow transplant in MPS I.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13441号 / 論医博第2240号 / 新制||医||1054(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 滝田 順子, 教授 江藤 浩之 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23782号 / 医博第4828号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 波多野 悦朗, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM