Runx1 is a central transcription factor in the development of the murine haematopoietic system and in the emergence and specification of its main key component, the haematopoietic stem cell. Previous studies suggested a requirement for Runx1 in a window of time stretching from mesoderm specification (E6.5) to mid-gestation (E11), but these studies did not investigate each primary haematopoietic site separately. During this PhD project, a Runx1 reactivatable knockout mouse model was used to study the impact of the absence of Runx1 from E9.5 to E11 in primary haematopoietic sites on early precursor populations, especially PreHSC Type I and II. At E9.5, the KO conceptus was already developmentally retarded, lacking progenitors and PreHSC Type II but was not devoid of PreHSC Type I, as demonstrated by flow cytometry, thus suggesting a requirement for Runx1 in the transition from PreHSC Type I to PreHSC Type II stage. Using a novel culture system that enables the potent in vitro maturation of precursors of HSCs into fully mature adult-repopulating HSCs, it was found that maturation of PreHSC Type I into HSCs was hindered in KO tissues, despite the expression of Runx1 in OP9 niche compartment, thus pointing towards a cell autonomous requirement for Runx1. In this model, the Runx1 allele was subsequently reactivated to a functional state by tamoxifen-induced Cre-mediated recombination (CreERT2 system). Tamoxifen / Cre toxicity on HSC maturation was evaluated during AGM reaggregate culture to achieve the best balance between the highest recombination levels and the lowest toxicity when Cre was induced in cell suspension prior to reaggregation. It was found that reactivation of Runx1 at E9.5 in primary haematopoietic sites was not sufficient to rescue haematopoietic development, thus suggesting a requirement for Runx1 before E9.5.

Liver fibrosis is a wound healing process in liver with¡@chronic injury and is characterized by the excess production and accumulation of extracellular matrix (ECM) component. Liver injury of any etiology may lead to activation of hepatic stellate cells (HSCs), which are trans-differentiated from lipocyte-like cells to highly proliferative myofibroblast-like cells. Activation of HSCs is considered a crucial event that promotes increased ECM production and consequently hepatic fibrosis. Liver fibros is resulted from a net increased synthesis and decreased degradation of ECM proteins. Pluchea indica (Less) has been reported to have antipyretic, anti-ulcer, anti-inflammatory, anti-oxidant, diuretic and anti-amoebic activities. Our previous studies showed that the aqueous extract of roots from P. indica (PIRAE) showed that it can suppress the growth and migration of HeLa and GBM8401 cancer cell lines, and also significantly reduce serum glutamate pyruvate transaminase (GPT), alpha-smooth muscle actin (£\-SMA) and collagen type I expression in animal model of liver fibrosis induced by thioacetamide (TAA). In this study, we plan to investigate the effects of PIRAE on activation, proliferation and migration of rat culture activated HSCs. The results indicated that protein expression of £\-SMA and collagen type I of HSCs was decreased followed by treatment of either 0.5 or 1.0 mg/ml PIRAE for 48 hours. In addition, the effects of PIRAE on proliferation in culture activated HSCs were assessed by analyses of cell growth curve, MTT, WST-1 and BrdU, respectively. The results showed that PIRAE inhibited HSCs proliferation in a dose- and time-dependent manner. Moreover, wound healing assay and transwell assay showed that PIRAE prevented migration in activated HSCs. In conclusion, PIRAE may suppresse culture activated HSCs proliferation, migration, and activation of culture activated HSCs, as well as accumulation of collagen type I.

For over 50 years, bone marrow transplants have used CD34 to select stem cells. Recent research suggests that the most primitive hematopoietic stem cells (HSCs), long-term HSCs (LT-HSCs), are found in the CD34-negative portion of murine and human bone marrow cells. LT-HSCs are rare and cannot be isolated directly, making them difficult to study. During a bone marrow transplant, these stem cells must find their way to the bone marrow niche and engraft to become blood cells. Several cell adhesion molecules on the stem cell engage with their ligands on the endothelial cells lining the bone marrow vasculature to control this migration. Human LT-HSCs cells do not migrate and engraft well when infused in vivo, which may be due to a lack of adhesion molecules. Thus, the goal of this study was to determine whether this population of HSCs lacked adhesion systems (proteins and carbohydrate modifications) and, if so, to improve their migration and engraft ability by modifying key mechanistic steps in the adhesion cascade. Therefore, we investigated how distinct hematopoietic stem cell populations migrate to the bone marrow using adhesion mechanisms. This study represents the first direct analysis of adhesion molecules expression in LT-HSC and will potentially shed light on methods to optimally use these very valuable cells in the clinical bone marrow and cord blood transplants worldwide.

La méthylation de l’ADN est la marque épigénétique la plus connue. Elle consiste en l’ajout d’un groupement méthyle au niveau de la cytosine, produisant la 5-méthyl-cystosine (5mC). Cette réaction chimique est catalysée par des ADN méthyltransférases : DNMT1, DNMT3A et DNMT3B. Peu de choses sont connues concernant les changements de 5mC au cours des lignages cellulaires dans l’embryon et comment cette marque contribue à l’établissement ou au maintien de l’identité cellulaire. Nous avons cherché à mieux comprendre ces mécanismes en étudiant la 5mC dans deux lignages cellulaires : les cellules primordiales germinales (PGCs) et les cellules souches hématopoïétiques (HSCs). Nous avons généré les premiers méthylomes de ces cellules au cours de leur développement chez la souris. Chez les PGCs, nous avons mis en évidence l’existence de deux phases de reprogrammation de la 5mC. Une première phase entre E9,5 et E13,5, où le génome des PGCs se déméthyle et une phase de reméthylation entre E14,5 et E17,5, chez les gamètes mâles uniquement. Néanmoins, certaines régions, dont notamment les éléments transposables sont résistants à la vague de déméthylation. L’utilisation de souris conditionnellement, nous a permis de mettre en évidence l’implication des protéines DNMT1 et UHRF2 dans le maintien de la 5mC au niveau de ces séquences. Concernant les HSCs, nous avons mis en évidence qu’elles acquièrent un profil de 5mC qui leur est propre lors de deux phases. La première a lieu dès l’apparition des HSCs dans l’organisme tandis que l’acquisition de la signature hématopoïétique définitive se déroule à l’âge adulte dans la moelle osseuse. L’utilisation de souris conditionnelles, nous a permis de mettre en exergue l’implication de DNMT3A et DNMT3B dans la mise en place de ces profils, avec un rôle prépondérant de DNMT3B lors de la phase d’acquisition précoce et de DNMT3A lors du verrouillage de leur profil de 5mC. / The methylation of DNA is a well-known epigenetic mark. It consists in adding a methyl group to a cytosine producing the 5-methylcytosine (5mC). This is catalysed by the DNA methyltransferase (DNMT) family: DNMT1, DNMT3A and DNMT3B. Little is known about the changes in DNA methylation that follow lineage decisions in the embryo and how these contribute, establish or maintain cellular identities. We are addressing these questions using as a model the specification of mouse primordial germ cells (PGCs) and mouse hematopoietic stem cells (HSCs) in the mouse embryo. We generate the first genome-wide maps of 5mC during their development. These maps highlight two waves of DNA methylation in PGCs. The first one takes place between E9,5 and E13,5, where the genome demethylates while the second one corresponds to a remethylation phase only in male PGCs between E14,5 and E17,5. Nevertheless, some regions, notably the transposable elements, are resistant to this demethylation wave. We demonstrate the implication of DNMT1 and UHRF2 in maintaining the 5mC on these regions using transgenic mice presenting specific deletion in PGCs. In HSCs, the 5mC maps highlight two wave of DNA methylation. The first one correlates with the first appearance of the HSCs in early embryos while the second one corresponds to their migration to the bone marrow and seems to act as a definitive lock for their hematopoietic identity. Using transgenic mice presenting specific deletions in HSCs, we prove the implication of DNMT3A and DNMT3B in hematopoietic stem cells, with a major role in locking HSC identity of DNMT3B during the first wave and a DNMT3A during the second one respectively.

CD26/DPPIV is a dipeptidyl peptidase that cleaves and destroys a variety of substrates such as the chemokine SDF-1α, a chemokine expressed along bone marrow endothelium, which is essential for the recruitment of hematopoietic stem cells (HSCs) via binding with its receptor CXCR4 to the bone marrow. Thus, CD26 is thought to interfere with the second step, chemokine/chemokine receptor interactions, of the cellular migration paradigm. To further study the role of CD26 in the migration of HSCs, we screened several human leukemic cell lines to find a model cell line that expresses active CD26 and discovered that the pro-monocytic cell line, U937 was optimal for this purpose. U937 cells were used to optimize a variety of assays including an CD26 activity assay and transwell migration assay with and without the use of a CD26 inhibitor, Diprotin A. Then, we isolated short-term and long-term HSCs from the bone marrow of C57BL/6N mice using a combination of surface markers and a fluorescence-activated cell sorter. The expression levels of Step 2’s homing molecules were measured by FACS in both fractions of HSCs. Interestingly, we detected differences in the expression of CD26 between these two populations that may help explain the inability of long-term HSCs to migrate to the bone marrow. Thus, through the use of a CD26 inhibitor the long-term HSCS migration to the bone marrow could be enhanced, leading to a prolonged and efficient stem cell engraftment activity. Such studies are could help develop protocols to improve stem cell engraftment for patients suffering from hematological diseases such as leukemia.

Ca2+/Calmodulin-dependent protein kinase type II (CaMK-II) is a Serine/Threonine protein kinase that is activated by Ca2+ and Calmodulin to phosphorylate substrates involved in myriad developmental processes. This project implicates CaMK-II in specification of HSCs, and zebrafish provide an ideal embryonic model to study hematopoiesis. Zebrafish genetic manipulation was achieved through: incubation in chemical inhibitors; injection of notochord-targed WT and DN CaMK-II constructs with Transposase; and injection of camk2g1 translation-blocking morpholino antisense oligonucleotide (MO). Whole-mount in situ hybridization (WISH) and immunolocalization on zebrafish embryos allowed visualization of key HSC markers and pathway components that implicated CaMK-II in the specification of HSCs. CaMK-II is a negative regulator of shh expression during HSC specification, but CaMK-II does not influence Shh during its well-documented role in vasculogenesis. CaMK-II appears to affect the spatial distribution of Shh protein, which accumulates near the notochord source and differentially affects expression of Shh target genes based on their distance from the notochord. This project also identifies the specific timing requirement for CaMK-II during HSC specification, as inhibition of CaMK-II consistently reduces HSC specification, but only if administered before 18hpf. CaMK-II also downregulates ezh2 in the DA during the time of HSC specification, and the Ezh2 inhibition rescues the loss of HSCs, suggesting that CaMK-II regulates the secretion of Shh from the notochord to epigenetically regulate expression of key HSC specification genes in the DA through EZH2 methyltransferase.

Haematopoietic stem cells (HSCs) emerge during embryogenesis and maintain hematopoiesis in the adult organism. Qualitative and quantitative assessment of HSCs can only be performed functionally using the in vivo long-term repopulation assay. Due to the lack of such data, little is known about the development of HSCs in the human embryo, which is a prerequisite for the development of new therapeutic strategies. Employing the xenotransplantation assay, I have performed here the spatio-temporal mapping of HSC activity within the human embryo and have shown that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region, specifically in the ventral wall of the dorsal aorta, and only later appear in the yolk sac, liver and placenta. Human AGM region HSCs transplanted into immunodeficient mice provide long-term high-level multilineage haematopoietic repopulation. These cells, although present in the AGM region in low numbers, exhibit a very high self-renewal potential. A single HSC derived from the AGM region generates around 600 daughter HSCs in primary recipient mice, which disseminate throughout the entire recipient bone marrow and are retransplantable. These findings highlight the vast regenerative potential of the earliest human HSCs and set a new standard for in vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine. I have also established a preliminary immunophenotype of the earliest human HSC. These data will be useful for my future studies on the mechanisms underlying the high potency of human embryonic HSCs and on the characterisation of embryonic HSC niche.

Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20216号 / 理博第4301号 / 新制||理||1618(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 細川 暢子, 教授 高田 彰二, 教授 七田 芳則 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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