Haematopoietic stem cells (HSCs) are capable of differentiation into all mature haematopoietic lineages, as well as long-term self-renewal and are consequently able to sustain the adult haematopoietic system throughout life. Currently, in the mouse, HSCs are understood to first appear in the aorta-gonad-mesonephros (AGM) region at embryonic day 11 via a process of maturation from precursors (pre-HSCs). This maturation within the AGM region involves the complex interplay of signalling between cells of the niche and maturing precursor cell populations, but is relatively little understood at a molecular level. Recently our understanding of the AGM region has been refined, identifying the progression from E9.5 to E10.5 and the polarity along the dorso-ventral axis as clear demarcations of the supportive environment for HSC maturation. In this thesis, I investigated the molecular characteristics of these spatio-temporal transitions in the AGM region through the application of RNA-sequencing. This enabled the identification of molecular signatures which may underlie the supportive functionality of the niche. I further compared these expression signatures to the transcriptional profile of an independent cell type, also capable of supporting HSC maturation, the OP9 stromal cell line. By combining this transcriptional information with an ex vivo culture system, I screened a number of molecules for their ability to support HSC maturation from early precursors, leading to the discovery of a novel regulator of HSC maturation: BMPER. Further characterisation of this molecule enabled the identification of its specific cellular source and the proposal that through its action as an inhibitor of BMP signalling it facilitates the maturation of precursors into HSCs. These results lend further detail and support to the role of BMP signalling in the regulation of HSC maturation as well as demonstrating the potential of these transcriptional profiles to yield novel mechanistic insight.

The potential of embryonic stem (ES) cells to generate any developmental or adult cell type holds much promise for regenerative medicine and in vitro modelling of development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages of the blood throughout adult life and are essential for the treatment of a vast number of haematalogic disorders. Current sources of HSCs for clinical use and research, including adult bone marrow, peripheral blood stem cells and umbilical cord blood, are limited by the number of HSCs they contain and by the availability of a suitable donor. A system that generates a reliable source of HSCs from ES cells would therefore be an ideal alternative. While much progress has been made in the generation of downstream lineages of the haematopoietic system, progress in the derivation of HSCs capable of long-term self-renewal and multilineage reconstitution from ES cells has been limited. Understanding of the developmental steps leading to HSC emergence in the embryo has been advancing in recent years. In particular, precursors of HSCs (preHSCs) have been isolated from the mouse embryo, characterised and matured into HSCs ex vivo using the specialised conditions of aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised that application of the aggregate culture system in the differentiation of ES cells could provide a missing link in the in vitro generation of HSCs. Here I have developed a novel ES cell differentiation system that employs the specialised conditions of the aggregate culture system, after an initial stage of mesoderm differentiation. I show that this system creates an environment for efficient haematopoietic and endothelial progenitor formation and generates cells of a preHSC type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+) surface phenotype. Notably, the system gives rise to cells that achieve low-levels of haematopoietic repopulation in sublethally irradiated NSG mice. The low-level repopulating cells persist for over 4 months in animals and show both myeloid and lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell differentiation does not improve levels of ES-derived repopulation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23794号 / 医博第4840号 / 新制||医||1058(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 河本 宏, 教授 小川 誠司, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Patients with Acute Myeloid Leukaemia (AML) present with the signs and symptoms of bone marrow failure. This finding spans the genetic and phenotypic diversity of the disease. The mechanism which underlies it is poorly understood. This thesis explores the effect of AML on the normal haematopoietic stem cell (HSC) population, using primary human diagnostic bone marrow samples. Previous work from our group suggested that AML induces a state of quiescence in HSCs, producing a differentiation block responsible for the observed cytopenias1. Reversal of this process might offer an alternative to the current treatment of patients with palliative transfusions. I have developed a flow cytometry-based technique to differentiate normal HSCs from leukaemia cells, selecting cells with the CD34+38-ALDHhighCLL1- expression signature. Validation of this technique by assessment of sorted cells by FISH and PCR, suggests it is successful in 73% of AML samples. In a further 25% of samples, it selects for a population significantly enriched for normal HSCs. We used this panel to investigate the concentration of HSCs at AML diagnosis, compared to controls. We show that there is no significant difference between HSC concentration at AML diagnosis (n=38, median [HSC] 2.5 cells/μl) and controls (n=24, median [HSC] 2.4 cells/μl). HSC concentration was not significantly affected by AML karyotype, patient age or gender. However, those patients presenting with a low HSC concentration at diagnosis (< 0.1 HSC/μl) were found to have a significantly worse outcome both in terms of overall and relapse-free survival, an effect apparently independent of age, gender and underlying karyotype. HSC concentration at diagnosis with AML may therefore represent a new independent prognostic marker. We then studied CD33 expression patterns on HSCs within Core Binding Factor mutated AML (n=37) at diagnosis, and found its expression to be significantly lower than on HSCs within controls (n=9) (17% versus 58%, p=0.005). CD33 expression on HSCs from AML samples rose significantly from diagnosis to remission (n=16) (17% to 58%, p=0.0001). This mirrors previous findings from our group using CD34low AML samples, and is, we believe, the first time that the antigenic signature of normal HSCs has been shown to be modified. 6 by the presence of AML. However, an in vitro assay to test the significance of these changes in terms of the cytotoxicity of GO towards normal HSCs did not demonstrate a significant difference between HSC subgroups. Finally, we attempted to investigate the mechanism by which AML might induce HSC quiescence by studying the comparative transcriptomes of HSCs from CD34low AML (n=6) and controls (n=6) by RNA-Seq, using direct cell to cDNA synthesis, followed by amplification. A first attempt resulted in poor quality data, with a significant proportion of reads mapping to non-coding DNA regions. A repeat approach, using utilising immediate RNA extraction post sorting resulted in significantly better quality data Bioinformatics analysis revealed differential expression of 6 genes between the 2 datasets (GNPDA1, ADGRG3, MIAT, WDR31, RP11-244H3.1 and RXFP1). GO enrichment studies using David highlighted a number of pathways including the TNF signalling pathway (p=0.003; after Benjamini-Hochberg correction p=0.51). Validation of these findings by independent qPCR, and functional exploration of enriched signalling pathways remains outstanding.

In adults blood stem cells, called haematopoietic stem cells (HSC), give rise to all blood cells throughout life. The origin and biology of HSCs during embryo development has been an intensely studied topic. Definitive HSCs are generated intra-embryonically in the aorta-gonad-mesonephros (AGM) region of the mid-gestation embryo. Recent research revealed that HSCs emerge through multistep maturation of precursors: proHSC → preHSC I → preHSC II → definitive HSC (dHSC). A hallmark of the HSC emergence is the appearance of intra-aortic haematopoietic clusters that are considered to be sites of haematopoiesis. It was shown in vitro that the E11.5 HSCs are slowly cycling compared to progenitor cells. However, cell cycle status and its role during early HSC development remain unclear. Here I used Fucci transgenic mice that enable in vivo visualisation of the cell cycle. Functional and phenotypic analysis showed that in the early embryo the proHSC precursors cycle slowly, whereas committed progenitors are actively cycling. Meanwhile the preHSC I precursors arising in the E10.5 AGM region become more rapidly cycling. They are located closer to the luminal cavity of the dorsal aorta, while their ancestors, the proHSCs, are slowly cycling and are located at base of the clusters. Furthermore, in the mid-gestation embryo the preHSC I become slowly cycling and are closer to the endothelial lining of the aorta, while they give rise to the actively cycling preHSC II that are located to the luminal area of the artery. Finally, definitive HSCs are mainly slowly cycling at this stage like their foetal liver counterparts. As expected, HSCs in adult bone marrow are mainly dormant. The data suggest that transition from one precursor type to another is accompanied by distinct changes in cell cycle profile and that HSCs become progressively quiescent during development. To test the role of cell cycle in HSC maturation, we used inhibitors against signalling pathways known to play important roles in HSC development. Notch inhibitor affected the cell cycle status of haematopoietic precursors, by possibly promoting them to rapidly proliferate and potentially blocking the maturation from preHSC I to preHSC II precursors. Shh antagonist had the opposite effect and enhanced the HSC activity from the preHSC I precursors. Altogether these results suggest that the cell cycle status plays an important role in the HSC development. A better understanding of the molecules that control this process will allow us to optimize the culture condition for generation of functional HSCs in the laboratory.

Philosophiae Doctor - PhD / Epidemiological studies have highlighted that leukaemia can be considered as the most prominent malignancy after radiation exposure during childhood. The lifetime risk on radiation-induced leukaemia for a given dose is 3 – 5 times higher for children compared to adults. The high risk at a young age is related to the elevated sensitivity of the red bone marrow where haematopoietic stem and progenitor cells (HSPCs) are located. HSPCs self-renewal capacity and long-life span increase their susceptibility to DNA damage accumulation, making them a major target of radiation-induced carcinogenesis. Proton beam therapy (PBT) is increasingly used to treat paediatric brain tumours due to its dose sparing properties compared to conventional X-ray based radiotherapy. However, concerns regarding the carcinogenic potential of secondary neutrons produced during PBT, especially in terms of their effect on HSPCs harboured in the cranial bone marrow of paediatric patients, remain. In this study, the radiobiological differences between 60Co γ-rays and p(66)/Be(40) neutron exposure was investigated to resolve the underlying mechanisms for the high radiosensitivity of HSPCs (CD34+ cells) isolated from umbilical cord blood (UCB). For both radiation qualities, an apparent dose-dependent increase in the frequency of radiation-induced MN was observed in CD34+ cells. Furthermore, increased cytogenetic damage was observed with the CBMN assay after neutron irradiation, which highlights its leukaemogenic potential. In addition, no difference was observed in the nuclear division index of the CD34+ cells post-irradiation between both radiation qualities. The number of DNA DSBs was assessed by microscopic scoring of γ-H2AX foci, 2 and 18 hours after radiation exposure. A significant higher number of DNA DSBs were observed 2 hours after neutron irradiation with 0.5 Gy, but decreased to similar levels for both radiation qualities after 18 hours. Different stages of apoptosis in CD34+ cells were studied at 18 and 42 hours numerous time points post-irradiation by flow cytometry using the Annexin/PI assay. In contrast to the γ-H2AX foci results, a significant difference in late apoptosis was observed at 18 hours and 42 hours between the two radiation qualities. The results point towards a fast error-prone DNA repair in HSPCs after neutron irradiation, which might contribute to genomic instability and leukemogenesis. In the second phase of the PhD project, the impact of age on radiosensitivity was investigated by comparing newborn T-lymphocytes with adult peripheral blood (APB) T-lymphocytes. The major difference between UCB and APB T-lymphocytes, is their immunophenotypic profile. Since it is known that different T-lymphocyte subsets have a difference in radiosensitivity, the fraction of CD4+, CD8+, naïve (CD45RA+) and memory (CD45RO+) T-lymphocytes was determined via flow cytometry in the two groups. The cytokinesis-block micronucleus (CBMN) assay was used to determine the extent to which age influences the frequency of cytogenic damage in response to 60Co γ-rays radiation. For both APB and UCB, an outspoken dose-dependent increase in the frequency of radiation-induced MN was observed at 0.5, 1, 3 and 4 Gy. However, no significant difference was observed at 4 Gy when comparing MN yields of APB and UCB. An increased radiosensitivity of newborn to adult donors of 34%, 42%, 29%, 26% and 16% was observed based on the MN scoring at doses of 0.5, 1, 2, 3 and 4 Gy, respectively. The lowest radiosensitivity was identified at the highest dose, which might explain the non-significant difference at 4 Gy. In addition, there was a clear trend that females were more sensitive to 60Co γ-rays radiation than males in both adults and newborns, even though the difference was not significant. The immunophenotypic study revealed that that both the CD4+ and CD8+ T-lymphocytes of newborns are mainly naïve. This is illustrated by the co-expression of CD45RA+ on 90.70% (range: 80.80% – 98.40%) and 95.90% (range: 89.60% – 98.80%) of CD4+ and CD8+ cells respectively. The composition of adult T-lymphocytes, in contrast, is clearly different with a more equal distribution between CD45RA+ and CD45RO+ subpopulations. This finding demonstrates that there are differences in the radiosensitivity between newborn and adult T-lymphocytes which might be linked to the immunophenotypic change of T-lymphocytes with age.

Identifying the sites and mechanisms involved in haematopoietic stem cells (HSCs) during development would improve our understanding of how to induce HSCs from alternative sources like embryonic stem cells, while offering insight into pathways involved in HSC-related diseases such as leukaemia. Adult-type HSC, or long-term reconstituting HSCs (LTR-HSCs), are widely defined as cells capable of reconstituting the entire haematopoietic system of a lethally irradiated adult recipient. The first LTR-HSCs emerge and expand in the aorta-gonad-mesonephros (AGM) region of the mid-gestation mouse embryo. Recently, the development of a novel reaggregate culture system has provided a valuable tool to identify key cell populations involved in LTR-HSC development. This system allows the mechanical dissociation of the E11.5 AGM region prior to culture whilst maintaining its ability to autonomously expand LTR-HSCs. Here, I show that reaggregate LTR-HSCs are CD45+Sca1+c-kit+CD31med and that IL-3, SCF, and Flt3l are required in order to achieve an optimal 150 fold LTR-HSC expansion. I also characterise the pattern of Runx1 expression in the adult and E11.5 AGM region of our novel Runx1EGFP reporter mouse and identify a population of EGFP+CD45-VE-cadherin- cells in the E11.5 AGM region that disappears during reaggregate culture. Finally, using the E11.5 AGM reaggregate culture, I show that while uro-genital ridges are potentially required for optimal LTR-HSC expansion, most LTR-HSCs are derived from the dorsal aorta (Ao) region, and that the dorsal aspect of the dorsal aorta (AoD) can contribute to the reaggregate LTR-HSCs compartment.

Mucopolysaccharidosis type IIIA (MPS IIIA) is caused by mutations in the N-sulphoglucosamine sulphohydrolase (SGSH) gene, leading to cellular accumulation of heparan sulphate and progressive neurodegeneration in patients. One of the proposed treatment methods is haematopoietic stem cell (HSC) gene therapy, which should result in an excess of SGSH produced in the peripheral organs and brain. The pre-clinical feasibility of this approach was demonstrated by our group in a mouse model of MPS IIIA. However, the overall efficiency of this method was limited and a number of approaches to solving these issues were addressed in this project in order to bring this therapy closer to clinical application. Our first aim was to optimise transduction of HSCs using cytokines, bovine serum albumin (BSA), and chemicals, such as MG132, genistein and valproic acid. Addition of BSA with cytokines improved cell viability, addition of MG132/ BSA/ cytokines improved transduction, but also caused cellular toxicity, while addition of genistein was inefficient. Addition of valproic acid with cytokines resulted in increased number of colony forming units. Next, we generated clinically applicable third generation pCCL lentiviral vector backbones with the eGFP reporter gene driven by one of ubiquitous hPGK or myeloid specific hCD11b and hCD18 internal human promoters, and optimised production of lentiviral vectors to increase titre and reduce production cost. These lentiviral vectors were used to transduce lineage depleted HSCs and transplanted into WT mice. Full chimerism and over 80% transduction were achieved with an average of 5 vector copy numbers/ cell. The hCD11b promoter resulted in the highest eGFP expression in monocytes and B cells in blood, but was weaker than the hPGK in T cells. The hCD18 promoter was more monocyte-specific but weak. Significant numbers of GFP-positive microglial cells were present in the brain from all groups, with an average of 25% transduced CD11b-positive cells in perfused mice. We subsequently codon-optimised (CO) the SGSH gene significantly improving enzyme activity, and transduced lineage depleted WT cells with one of hCD18.SGSH-CO, hCD11b.SGSH-CO, or hPGK.SGSH-CO lentiviral vectors, or MPS IIIA cells with either hCD11b.SGSH-CO or hPGK.SGSH-CO lentiviral vectors. These transduced cells were transplanted into MPS IIIA mice and outcomes were measured 6 months later. Only treatment with the hCD11b.SGSH-CO-LV transduced WT or MPS IIIA HSCs corrected abnormal behaviour of MPS IIIA mice. However, all treatments resulted in complete GAG storage clearance in the periphery and brain, and significantly elevated enzyme activity in the brain, liver and spleen to 7-11%, 60-75%, and 170-250% of WT enzyme activity respectively. A fine threshold of over 8.6% brain enzyme activity appeared to be required for behavioural correction in MPS IIIA mice. Further assessment of treated mice for the amount of secondary storage, HS sulphation patterning, neuroinflammation and longevity are still required for complete therapeutic assessment. However, it appears that neurological correction of the MPS IIIA mouse using MPS IIIA cells is feasible using a clinically-relevant pCCL vector with the hCD11b promoter and the codon-optimised SGSH gene.