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<p>The generation of hematopoietic progenitors from human pluripotent cell sources for use in personalized medicine is an attainable goal for the ease of clinical intervention using these cells. Furthermore, generated platelets and mature red blood cells are enucleated which allows for the use of induced pluripotent stem cells as a starting source or other sources of genetic manipulation. Generating these cells has proven difficult as the cells appear to be stuck in a primitive state of differentiation and do not mature into an adult phenotype. This thesis shows that inhibition of the hedgehog signaling pathway early in the differentiation of pluripotent stem cells induces a maturation towards definitive hematopoiesis. Generated erythroid cells were shown to express beta globin at the transcript as well as protein level. This maturation effect was confirmed to occur through central hedgehog repressor, Gli3R, through genetic manipulation. Further interrogation of this mechanism showed that globin regulation was not mediated by chromatin methylation by the polycomb repressive complex. Finally, Gli3R was also shown to not act as a transcription factor influencing globin expression directly and is therefore engaging separate regulatory mechanisms. This data provides great strides towards the generation of clinically relevant hematopoietic populations from pluripotent sources, however Gli3R’s direct mechanism of action remains to be determined.</p> / Master of Science (MSc)

Hematopoiesis is the highly regenerative process of producing billions of blood cells each day, including white blood cells, red blood cells, and platelets. Given the relatively short life span of these mature cells, hematopoiesis is dependent on stem and progenitor cells to generate renewed progeny, which represents a tightly regulated process. This includes cell intrinsic and external factors, and where dysregulation can lead to anemia and cancer. As such, the hematopoietic hierarchy has been intensely studied for nearly a century and represents a gold standard model of cell fate and developmental biology, in research and clinical applications. Cellular models, such as in vitro culture and human-mouse xenografts in vivo, have been developed to explain complex phenomena pertaining to hematopoiesis and also interrogate processes which are too invasive to study in humans. Hematopoietic generation is required beyond sustaining homeostasis, and progenitors can be damaged through cytotoxic injuries such as radiation and standard chemotherapy, and also undergo leukemic transformation. There are two main treatment modalities for leukemia patients (a) receiving a stem cell transplant, and (b) drug or radiation-based therapy. In the former, shortages of donors and stem cells has remained an unmet clinical need for decades. In the latter, selective targeting of genetic mutations has become a successful standard-of-care in leukemias such as chronic myelogenous leukemia and acute promyelocytic leukemia. However, in the most common adult hematologic malignancy, chronic lymphocytic leukemia (CLL), similar targeting therapies have not been developed. Altogether, shortages of stem cells from healthy donors, chemotherapy-induced immune dysfunction, and a lack of targeted therapies, all reinforce the immediate need for innovative cellular models to address these clinical problems. To generate additional sources of human hematopoietic progenitors for laboratory study, human PSCs have been used. Unlike hematopoietic progenitor cells collected from healthy and leukemic donors, human pluripotent stem cells (PSC) can be easily propagated and expanded in vitro. PSCs can generate hematopoietic progenitor cells, but they remain poorly understood and have not been robustly applied to solve the aforementioned deficiencies related to patient treatment. Importantly, the biological regulation of both hematopoiesis and PSCs has been experimentally confirmed to significantly deviate between humans and other animals, such as mice, further reinforcing the importance of human-specific cell models of hematopoiesis. Therefore, I hypothesized that human stem cell models provide a focused approach to interrogate the regulation of hematopoiesis from the apex of the hierarchy, which can be used to understand the promotion of healthy hematopoiesis and understand malignant transformation. Collectively, the data presented within this thesis offer a deeper conceptualization of human stem cell models and the deconvolution of several complex components of hematopoietic regulation. This work has revealed novel, clinically relevant, and actionable targets to ultimately enable the promotion of healthy hematopoiesis on multiple fronts. / Thesis / Doctor of Philosophy (PhD) / This thesis presents research on novel molecular and genetic regulatory pathways of self-renewal and differentiation in models of healthy and malignant human hematopoiesis. The origin of healthy hematopoietic regulation stems from a large body of work spanning decades and encompasses many efforts by others to derive hematopoietic stem cells from human pluripotent cells. The development of a genetic model for the malignant regulation of CLL was truly serendipitous, was propelled through robust and intriguing results that begged for further exploration, and filled a clinical gap in identifying actionable targets in CLL. Lastly, these two projects, along with my supportive roles in other published works throughout my graduate studies, instructed me to develop a human-mouse transplant model to uncover the biology of regenerating healthy hematopoiesis during injury.

The development of the cardiovascular system, including the heart and circulating blood within a vascular network, relies on mesoderm-derived cells to contribute to the development of both cardiac and hematopoietic tissues. This dissertation focuses on exploring the roles of crip2, downstream of the transcription factor Nkx2.5 established from an RNA sequencing dataset, in cardiac and hematopoietic development using the zebrafish model. In Chapter 2, we investigate the influence of Crip proteins on the development of the zebrafish heart. Congenital heart defects (CHDs), affecting approximately 1% of live births, arise from structural anomalies during heart development primarily caused by genetic mutations. While there isn’t just one driver of CHDs, transcription factors such as Nkx2.5, play a pivotal role in guiding cardiac morphogenesis. NKX2-5-associated CHDs often involve outflow tract (OFT) malformations. The development of the heart involves two progenitor cell populations, the first heart field (FHF) and second heart field (SHF), contributing to the linear heart tube and subsequent growth. Despite understanding the role of Nkx2.5, the spatiotemporal mechanisms directed by Nkx factors in SHF progenitor specification, proliferation, and identity maintenance remain elusive. This study aims to uncover novel effectors of Nkx transcriptional regulation, using RNA sequencing on dissected wild-type and nkx2.5-/- zebrafish hearts at 26 hours post fertilization (hpf). This work focuses on a LIM domain protein, cysteine rich intestinal protein 2 (crip2), identified as a mis-regulated gene in nkx2.5-deficient embryos, and we explore its role downstream of nkx genes in SHF-derived arterial pole formation. While crip2 is abundantly expressed in the developing heart, the family member crip3 also shows a mild expression pattern. Loss-of-function mutations in crip2 and crip3 (referred to as cripDM) reveal normal cardiac chamber specification. Atrioventricular canal morphology remains unaffected in cripDM embryos. The OFT in cripDM embryos displays a significant dilation, accompanied by increased ltbp3 expression. Surprisingly, the smooth muscle cell population of the OFT does not explain the size increase. This research expands our understanding of OFT development, highlighting the multi-layered contributions of various cell types and factors. In Chapter 3, we further examine the role of crip2 in the development of hematopoietic stem cells given its endothelial expression pattern. Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that crip2 has a previously unrecognized function in establishing the proper endothelial cell environment for HSPC specification. CripDM embryos exhibit decreased emergence of HSCs by 26 hpf. Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineage production between 30 -72 hpf; these perturbations in the hematopoietic lineages recover by 96 hpf. To decipher the spatiotemporal mechanisms underlying the cripDM phenotype, we performed single cell RNA (scRNA) sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and mis-regulation of Notch signaling pathways in the cripDM embryos. Building on these data, our ongoing studies aim to investigate how crip2 regulates the endothelial niche of the ventral aorta to produce HSCs early in definitive hematopoiesis. We anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

HHT wird in verschiedene Typen unterteilt, wobei Typ 1 am häufigsten vorkommt und durch vaskuläre Unregelmäßigkeiten und Blutungsepisoden aufgrund von CD105-Haploinsuffizienz gekennzeichnet ist. Da CD105 vor allem in ECs exprimiert wird, nutzte unsere Gruppe das VE-cadh-ERT2:cre-CD105f/f-Mausmodell, um die Auswirkungen des EC-CD105-Mangels auf das vaskuläre und hämatopoetische System des Knochenmarks von erwachsenen Mäusen zu untersuchen. Dieses Modell schuf eine Umgebung, in der ECs in erwachsenen Mäusen mit zuvor regulärer CD105-Expression in einer In-vivo-Umgebung reduzierte Werte aufwiesen, was zum ersten Mal eine Zeitraffer-Analyse der EC-Umstrukturierung als Reaktion auf CD105-Mangel ermöglichte. In der vorliegenden Studie untersuchten wir das Zusammenspiel zwischen Blutgefäßumbau und hämatopoetischen und Immunzellaktivitäten nach EC-CD105-Mangel. Insbesondere konnten wir einen zeitabhängigen vaskulären Umbauprozess im Knochenmark feststellen, der etwa vier Wochen andauerte. Dieser Umbau war durch eine allmähliche Abnahme der Größe einzelner Blutgefäße gekennzeichnet, die in Woche 4 zum Stillstand kam. Bemerkenswert ist, dass diese Schrumpfung mit Veränderungen des EC-Zyklus und der Lebensfähigkeit einherging. Insbesondere waren die ECs von cKO-Mäusen in den Wochen 2 und 3 weniger ruhig und in Woche 4 nach der Induktion des EC-CD105-Mangels weniger lebensfähig. Bemerkenswert ist, dass der Anstieg der Proliferation in Woche 2 mit einer vorübergehenden Zunahme der Bindung der VEGFR2-Gensignatur korrelierte. Neben diesen Veränderungen in der Proliferation und Lebensfähigkeit zeigten die ECs zeitabhängige Veränderungen in ihrer Adhäsions, MMP- und Integrin-Aktivität. Darüber hinaus wurden diese Veränderungen der Endothelaktivität von einer erhöhten Menge an Wachstumsfaktoren im extrazellulären Proteingehalt des Knochenmarks in Woche 2 begleitet, die in Woche 4 nicht mehr vorhanden waren, einschließlich VEGF-A. Nach intrakardialer Injektion von Brustkrebszellen wiesen Mäuse mit EC-CD105-Mangel 72 Stunden nach der Injektion weniger metastatische Knoten in ihrem Knochenmark auf als ihre WT-Wurfgeschwister, was darauf hindeutet, dass EC-CD105 ein wichtiger Akteur bei der Regulierung der Extravasation von Brustkrebs in das Knochenmark ist. Darüber hinaus stellt Woche 3 möglicherweise einen entscheidenden Zeitraum dar, in dem das Endothel des Knochenmarks anfälliger für die Infiltration verschiedener Zelltypen wird, wie der vorübergehende Anstieg von B-Zellen und Monozyten und der dauerhafte Anstieg der Neutrophilen- und Eosinophilenzahlen im Blutkreislauf zeigen. Darüber hinaus stieg die Extravasation von Dextran in das Knochenmark nach Woche 3 ebenfalls dauerhaft an. Bemerkenswert ist, dass die Zahl der Eosinophilen und Neutrophilen auch nach heterozygoter Deletion von EC-CD105 anstieg, während die Zahl der Monozyten und B-Zellen davon unberührt blieb. Als Teil der Auswirkungen des homozygoten EC-CD105-Mangels wurde die Sequestrierung von Erythrozyten und PLTs durch die Milz erhöht, was zu Splenomegalie und einem Rückgang der Erythrozyten und PLTs im Kreislauf führte. Gleichzeitig stieg die Zahl der HSPCs, die zu Erythrozyten und PLTs im Knochenmark führen, was darauf hindeutet, dass das Knochenmark die Produktion von Erythrozyten und PLTs als Ausgleichsmechanismus erhöht. Heterozygoter EC-CD105-Mangel führte auch zu einem dauerhaften Anstieg der Neutrophilen- und Eosinophilenzahlen im Blutkreislauf in Woche 3. Die Zahl der B-Zellen und Monozyten blieb jedoch unverändert. Dies korrelierte mit einem fehlenden Unterschied in der Größe der Blutgefäße in Woche 4. Dies deutet darauf hin, dass das Gefäßsystem des Knochenmarks nach einem homozygoten EC-CD105-Mangel aufgrund des Gefäßumbaus eine erhöhte Anfälligkeit für die Infiltration von B-Zellen und Monozyten aufweist. Darüber hinaus führte die heterozygote EC-CD105-Deletion nicht zu signifikanten Unterschieden in der Anzahl der Erythrozyten und PLT im Blutkreislauf. Dieser fehlende Unterschied ging einher mit einer fehlenden Splenomegalie bei diesen Mäusen und keinen signifikanten Unterschieden in der Anzahl der HSPCs, die zu Erythrozyten und PLTs in ihrem Knochenmark führen. Diese Ergebnisse unterstützen die Annahme, dass ein homozygoter EC-CD105-Mangel zu einer verstärkten Sequestrierung von PLTs und RBCs in der Milz und damit zu Splenomegalie führt.:Table of Contents Acknowledgements 3 1 INTRODUCTION 9 1.1 Endoglin (CD105) 9 1.2 The endothelium and angiogenesis 12 1.3 The hematopoietic niche 17 1.4 Vascular control of hematopoiesis 21 1.4.1 EC control of oxygenation 22 1.4.2 Endothelial secretory factors and the perivascular niche 22 1.4. Remodeling of the ECM 23 1.4.4 Physical barrier separating marrow and lumen 24 1.5 β-integrin family 25 2 Aims of this thesis 28 3 MATERIALS AND METHODS 30 3.1 Tamoxifen treatment 30 3.2 Blood analysis 30 3.3 Flow cytometry and cell sorting 30 3.4 Next generation sequencing 30 3.5 Bone isolation for histological analysis 31 3.6 Bone sectioning and microscopy image acquisition 31 3.7 Dextran injection 31 3.8 Dextran area analysis 32 3.9 Breast cancer homing to bone marrow 32 3.10 Blood vessel histological analysis and calculation of distribution peak 32 3.11 Flow cytometry 33 3.12 Cell cycle analysis with Ki67 33 3.13 Cell viability analysis with Annexin V 33 3.14 Extracellular integrin staining 34 3.15 Intracellular integrin staining 34 3.16 Proteome profiler 35 3.17 Statistics 35 3.18 Breast cancer homing to bone marrow 36 3.19 Micro-computed tomography 36 4 RESULTS 39 4.1 Induced CD105-deficiency in endothelial cells of the bone marrow reduces blood vessel size 39 4.2 EC-CD105 deficiency differentially affects circulating cell populations throughout time 41 4. 3 EC-CD105 deficiency reorganizes hematopoietic progenitors in the bone marrow 45 4.4 CD105 deficiency causes vessels to remodel through endothelial cell proliferation and consequent apoptosis 48 4.5 CD105 deficiency differentially switches EC activity from angiogenic to apoptotic in a time dependent manner 51 4.6 EC-CD105 deficiency reduces EC adhesion and regulates integrin dynamics in a time dependent manner 57 4.7 Partial EC-CD105 deficiency affects immune cell dynamics in a time dependent manner 61 4.8 EC-CD105 deficiency affects cancer homing to bone marrow 65 5 DISCUSSION 67 5.1 EC-CD105 deficiency induces temporally orchestrated vasculature remodeling in the bone marrow 67 5.2 EC-CD105 deficiency affects circulating cells and the hematopoietic niche 70 5.3 EC-CD105 deficiency affects EC activity in a time dependent manner, cascading into affecting EC-circulating cell dynamics 73 5.4 EC-CD105 deficiency impairs breast cancer metastasis to bone marrow 76 6.1 Summary 79 6.2 Zussamenfassung 81 Bibliography 83 Deklaration 96 List of abbreviations 99

Indiana University-Purdue University Indianapolis (IUPUI) / Adult hematopoietic stem cells (HSC) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment or self-renewal. We have demonstrated that mouse and human HSC express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSC to PGE2 enhances their homing, survival and proliferation, resulting in increased long-term repopulating cell and competitive repopulating unit (CRU) frequency. HSC pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon multiple serial transplantations, with full multi-lineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression and enhances their migration to SDF-1α in vitro and homing to bone marrow in vivo and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. While PGE2 pulse of HSC promotes HSC self-renewal, blockade of PGE2 biosynthesis with non-steroidal anti-inflammatory drugs (NSAIDs) results in expansion of bone marrow hematopoietic progenitor cells (HPC). We co-administered NSAIDs along with the mobilizing agent granulocyte-colony stimulating factor (G-CSF) and evaluations of limiting dilution transplants, assays monitoring neutrophil and platelet recoveries, and secondary transplantations, clearly indicate that NSAIDs facilitate mobilization of a hematopoietic graft with superior functional activity compared to the graft mobilized by G-CSF alone. Enhanced mobilization has also been confirmed in baboons mobilized with G-CSF and a NSAID. Increases in mobilization are the result of a reduction of signaling through the PGE2 receptor EP4, which results in marrow expansion and reduction in the osteoblastic HSC niche. We also identify a new role for cannabinoids, an eicosanoid with opposing functions to PGE2, in hematopoietic mobilization. Additionally, we demonstrate increased survival in lethally irradiated mice treated with PGE2, NSAIDs, or the hypoxia mimetic cobalt chloride. Our results define novel mechanisms of action whereby eicosanoids regulate HSC and HPC function, and characterize novel translational strategies for hematopoietic therapies.

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The main topic of my PhD studies was to investigate the role of sumoylation in the function of Ikaros transcription factor, that regulates the lymphocyte differentiation and function. Sumoylation is a posttranslational modification that can change the properties and regulate the function of a given protein. Up to now, one study addressed the question of how sumoylationmodulates Ikaros function. It shows that Ikaros is sumoylated in total primary thymocytes, and that this dynamic event modulates Ikaros' repressive function. It also describes two consensus sumoylation sites on Ikaros (K58 and K240), the sumoylation of which leads to loss of Ikaros repressive function in ectopic reporter gene assays. The final conclusion of the study is that sumoylation does not alter the nuclear localization of Ikaros but acts as a mechanism disrupting its participation in both histone deacetylase (HDAC) dependent and independent repression. My work shows the presence of additional sumoylation site on Ikaros and demonstrates that sumoylation does not significantly alter its interaction with the nucleosome remodelling and histone deacetylase (NURD) complex in T-cell lines. The functional analysis of sumo-deficientmutants indicates a complex role of this modification in regulating Ikaros' transcriptional properties. The identification of this new sumoylation site contributes to a better understanding of Ikaros' dual repressive - activating function and suggests the existence of conditional Ikaros' interacting partners. Moreover, the different Ikaros splicing isoforms would have differentsumoylation profiles, which would complete the knowledge of their functional diversity.