This thesis describes a highly interdisciplinary approach to discern the differing impact of scaffold dimensionality and physical space restrictions on the behavior of single cells. Rolled-up nanotechnology is employed to fabricate three-dimensional (3D) SiO/SiO2 microtube geometries of varied diameter, that after a biofunctionalization step are shown to support the growth of U2OS and six different types of stem cells. Cell confinement quantifiable through the given microtube diameter is tolerated by U2OS cells through a remarkable elongation of the cell body and nucleus down to a certain threshold, while the integrity of the DNA is maintained. This confinement for NSPCs also leads to the approaching of the in vivo morphology, underlining the space-restrictive property of live tissue. The dimensionality of the cell culture scaffold however is identified as the major determiner of NSPC migration characteristics and leads to a morphologically distinct mesenchymal to amoeboid migration mode transition. The 3D microtube migration is characterized by exclusively filopodia protrusion formation, a higher dependence on actin polymerization and adopts aspects of in vivo-reported saltatory movement. The reported findings contribute to the determination of biomaterial scaffold design principles and advance our current understanding of how physical properties of the extracellular environment affect cell migration characteristics.

Aging human brains are prone to neurodegenerative disorders, the most common being the Alzheimer’s disease (AD). Currently, there is no cure for AD, and patients progressively lose neurons leading to reduction in the brain mass. Humans cannot circumvent and counteract this disease. For instance, chronic inflammation that manifests through mild to late stages of the pathology cannot be resolved. The synaptic degeneration that underlies cognitive decline cannot be reversed. As a general outcome, neurons deteriorate and new neurons cannot replace the lost ones. This is in part due to reduced proliferative and neurogenic ability of neural stem cells (NSCs), which normally produce neurons, albeit rather a limited lineage. Recently, in AD patients, neurogenic outcome was shown to reduce dramatically (Moreno-Jimenez et al., 2019; Tobin et al., 2019). This lack of neurogenic input from NSCs in human brains is emerging as a new aspect through which we might find a chance to counteract AD. One prominent question is to find ways to re-activate our NSCs in pathology conditions. Zebrafish is known to have a remarkable regenerative ability enabling it to regenerate its brain as well. Zebrafish brain possesses several neurogenic regions that harbor NSCs to allow continuous neurogenesis throughout adulthood and during regeneration. Radial glial cells in the zebrafish brain act as NSCs that respond to neuronal damage by enhancing brain plasticity and initiating neuroregeneration. Special molecular mechanisms are involved in activating NSCs to form new neurons and initiate the regenerative response. In my PhD project, I aimed to identify such regenerationassociated molecular mechanisms in AD-like neurodegenerative conditions. To investigate the molecular programs that mediate regenerative response in neurodegenerative conditions, we first generated an amyloid-mediated neurodegeneration model in adult zebrafish to mimic certain pathophysiological aspects of AD. We used synthetic Amyloid-β-42 (Aβ42) peptides and injected into the zebrafish brain using cerebroventricular microinjection (CVMI) method. These peptides were tagged with robust cell-penetrating peptide, which were previously shown to efficiently deliver cargo molecules into the zebrafish brain. This approach led to an acute model of neurodegeneration in which Aβ42 deposition was prominent in neurons in adult zebrafish brain, and also exhibited phenotypes reminiscent of human AD5 pathophysiology: apoptosis, inflammation, synaptic degeneration, and cognitive deficits. In contrast to the mammals, zebrafish brain induced the NSC proliferation and enhanced the neurogenesis to initiate a regenerative response. To identify the mechanisms behind this response, we performed whole-RNA transcriptome analyses, which revealed that several genes associated with immune-related signaling pathways were significantly enriched. We further found that Interleukin-4 (IL-4) is activated primarily in neurons and microglia in response to Aβ42, and is sufficient to increase NSC proliferation and neurogenesis. IL-4 binds to its cognate receptor IL4R that is expressed in NSCs, and activates the downstream signaling cascade via STAT6 phosphorylation. These results indicate that Aβ42-induced neurodegeneration in adult zebrafish brain leads to regenerative response mediated by direct activation of NSCs through a neuro-immune cross talk mediated by IL-4 signaling via STAT6 phosphorylation. In an approach to further elucidate how IL-4 signaling would mediate the NSCs response, we performed another whole-RNA transcriptome analyses after IL-4 treatment in homeostatic brains. We found that, apart from direct activation of NSC proliferation, IL-4 also has an indirect effect on NSCs through factors secreted by neurons. Single-cell transcriptomics further revealed the heterogeneity of the NSCs pool in the zebrafish brain, which responds directly or indirectly to Aβ42-induced IL-4. We found that IL-4 induces NSC proliferation and subsequent neurogenesis by suppressing the tryptophan metabolism and reducing the production of the neurotransmitter Serotonin. NSC proliferation was suppressed by Serotonin via downregulation of brain-derived neurotrophic factor (BDNF) in Serotonin-responsive periventricular neurons. BDNF itself enhanced NSC plasticity and neurogenesis via NGFRA/NFkB signaling in zebrafish. This regulatory network is not active in rodents. With these results, we identified a novel IL-4-dependent molecular mechanism of NSC proliferation that is mediated by Serotonin-BDNF-NGFRA regulatory axis. Our results elucidated a novel crosstalk through neuron-glia interaction that regulates regenerative neurogenesis in adult zebrafish AD model. Additionally, we identified two functionally distinct populations of NSCs, which mediate NSCs plasticity through distinct gene expression profiles and versatile signaling mechanisms. Collectively, we propose that zebrafish serves as an excellent model to investigate regeneration-associated mechanisms that enables the inherent capacity of enhanced regenerative neurogenesis upon neurodegeneration. We found that specific signaling6 mechanisms are active in specific subtypes of NSC populations in adult zebrafish brain. Since these mechanisms are normally inactive in NSCs of mammalian brains, particularly in rodents after AD-like conditions, we speculate that activating such candidate mechanisms in distinct NSCs population in mammalian brains could induce NSCs plasticity response. Indeed, our studies also suggested that some of these candidates could be harnessed to force human NSCs to become proliferative and neurogenic. Therefore, my PhD work opened up a new avenue of research that utilizes zebrafish for understanding what it takes for a vertebrate NSC to remain neurogenic even after AD pathology. Overall, I believe that this research route will be instrumental in designing nature-inspired therapeutic strategies for AD in regenerative medicine.

The melanoma cell adhesion molecule defines mesenchymal stromal cells in the human bone marrow that regenerate bone and establish a hematopoietic microenvironment in vivo. The role of the melanoma cell adhesion molecule in primary human mesenchymal stromal cells and the maintenance of hematopoietic stem and progenitor cells during ex vivo culture has not yet been demonstrated. We applied RNA interference or ectopic overexpression of the melanoma cell adhesion molecule in human mesenchymal stromal cells to evaluate the effect of the melanoma cell adhesion molecule on their proliferation and differentiation as well as its influence on co-cultivated hematopoietic stem and progenitor cells. Knockdown and overexpression of the melanoma cell adhesion molecule affected several characteristics of human mesenchymal stromal cells related to osteogenic differentiation, proliferation, and migration. Furthermore, knockdown of the melanoma cell adhesion molecule in human mesenchymal stromal cells stimulated the proliferation of hematopoietic stem and progenitor cells, and strongly reduced the formation of long-term culture-initiating cells. In contrast, melanoma cell adhesion molecule-overexpressing human mesenchymal stromal cells provided a supportive microenvironment for hematopoietic stem and progenitor cells. Expression of the melanoma cell adhesion molecule increased the adhesion of hematopoietic stem and progenitor cells to human mesenchymal stromal cells and their migration beneath the monolayer of human mesenchymal stromal cells. Our results demonstrate that the expression of the melanoma cell adhesion molecule in human mesenchymal stromal cells determines their fate and regulates the maintenance of hematopoietic stem and progenitor cells through direct cell-cell contact.

Background Hematopoietic stem cells located in the bone marrow interact with a specific microenvironment referred to as the stem cell niche. Data derived from ex vivo co-culture systems using mesenchymal stromal cells as a feeder cell layer suggest that cell-to-cell contact has a significant impact on the expansion, migratory potential and ‘stemness’ of hematopoietic stem cells. Here we investigated in detail the spatial relationship between hematopoietic stem cells and mesenchymal stromal cells during ex vivo expansion. Design and Methods In the co-culture system, we defined three distinct localizations of hematopoietic stem cells relative to the mesenchymal stromal cell layer: (i) those in supernatant (non-adherent cells); (ii) those adhering to the surface of mesenchymal stromal cells (phase-bright cells) and (iii) those beneath the mesenchymal stromal cells (phase-dim cells). Cell cycle, proliferation, cell division and immunophenotype of these three cell fractions were evaluated from day 1 to 7. Results Phase-bright cells contained the highest proportion of cycling progenitors during co-culture. In contrast, phase-dim cells divided much more slowly and retained a more immature phenotype compared to the other cell fractions. The phase-dim compartment was soon enriched for CD34+/CD38− cells. Migration beneath the mesenchymal stromal cell layer could be hampered by inhibiting integrin β1 or CXCR4. Conclusions Our data suggest that the mesenchymal stromal cell surface is the predominant site of proliferation of hematopoietic stem cells, whereas the compartment beneath the mesenchymal stromal cell layer seems to mimic the stem cell niche for more immature cells. The SDF-1/CXCR4 interaction and integrin-mediated cell adhesion play important roles in the distribution of hematopoietic stem cells in the co-culture system.

Human senses are specifically designed to recognize and understand the world that surrounds us. Even though we have five senses, vision alone is responsible for at least 30 % of the sensory input to our brain. The visual process is initiated in a highly specialized cell type, the photoreceptors. These are light-sensitive cells located in the retina, a layered nervous tissue situated at the back of the eye. Retinal degeneration diseases are a highly heterogeneous group of conditions that include mutations affecting the survival, maintenance and proper functioning of photoreceptors or the adjacent retinal pigment epithelium (RPE). Such mutations, alone or in combination with environmental factors, cause the loss of the affected cells, and therefore, impairment of the visual sense. Retinitis Pigmentosa and Age-related Macular Degeneration are typical examples of retinal degenerative diseases eventually leading to blindness. In the first one, rod photoreceptors degenerate and consequently also cone photoreceptors are lost. The second is characterized by malfunction and loss of both, RPE and photoreceptor cells. Many current therapeutic approaches for the treatment of retinal degenerative diseases focus on slowing down the progression of the disease, rather than restoring the visual function. Currently, new therapies with the potential to recover the visual signal are under development. Some of these therapeutic strategies have already reached clinical stages, including gene therapy or retinal prosthesis. However, gene therapy approaches require the presence of remaining photoreceptors and, furthermore, particular targeting of disease-related genes. Retinal prosthesis still require improvement in terms of long-term biocompatibility and relevant visual function recovery. An alternative strategy for vision restoration is cell replacement of the lost photoreceptors, which is potentially suitable for targeting late stages of retinal degeneration diseases, independently of the inherent cause of the disease. Human vision relies primarily on cone photoreceptors, which are the cells responsible for color and high acuity vision under daylight conditions. However, cones represent a minority of the photoreceptors within the retina, and so, due to the low availability of these cells, cone photoreceptor transplantation studies lag behind rod transplantation studies. Consequently, in this study, strategies to increase the numbers of cone photoreceptors within mouse embryonic stem cells (mESC)-derived retinal organoids, which represent a potential cell source for transplantation studies, were explored. In this regard, I manipulated developmental pathways known to be involved in retinal development, such as Notch signaling, through the addition of various compounds in the retinal organoid maturation media. However, early cone markers have not yet been definitively identified, complicating the detection and isolation of cone photoreceptor precursors within the organoids. Therefore, a new early cone-reporter mESC line was generated in the course of this study as a valuable tool with the potential to facilitate the development of novel cone photoreceptor replacement therapies. Equally important in the field of photoreceptor cell replacement is the understanding of how the transplanted donor cells interact with the host retina. Previous studies have shown that visual function improvement is possible after transplanting rod or cone-like photoreceptor precursors into the sub-retinal space of mouse models for retinal degeneration. For many years it has been assumed that the underlying mechanism for the observed vision improvement was the migration and structural integration of donor cells into the host outer nuclear layer, where they mature and establish synaptic connections with the host retinal circuitry. However, experiments performed in this study demonstrate, for the first time, that upon transplantation donor and host photoreceptors exchange cytoplasmic material rather than structurally integrate into the host outer nuclear layer. Furthermore, insights into the transferred cytoplasmic content are given, i.e. that mRNA, but not mitochondria are exchanged by donor and host photoreceptors. This novel way of photoreceptor-photoreceptor communication led to a paradigm change in the field of retinal transplantation, requiring a re-interpretation of former transplantation studies. In addition, the discovery of the material transfer phenomenon might serve as a starting point for the development of novel therapeutic strategies based on cell-cell support for the treatment of retinal degenerative diseases. This study generated new knowledge in two important topics related to the development of cell therapies for retinal degeneration diseases, including the development of tools for cone transplantation studies as well as elucidating the interaction between donor and host cells upon transplantation.

Die Nicht-alkoholische Steatohepatitis (NASH), gehörig zu der Gruppe der chronischen Lebererkrankungen als eine schwere Form der Nicht-alkoholischen Fettleber-erkrankungen (NAFLD), nimmt in ihrer Prävalenz ständig zu. Gründe dafür sind u.a. eine gesteigerte Nahrungsaufnahme sowie Veränderungen der Nahrungszusammen-setzung. Es kommt zur Ausbildung einer Steatose, die sich unter Mitwirkung verschie-dener Einflussfaktoren zur Steatohepatitis weiterentwickeln kann, wobei die Pathoge-nese noch nicht genau verstanden ist. Die Nicht-alkoholische Steatohepatitis geht oft einher mit Insulinresistenz und starkem Übergewicht. Die Folgen für die Leber sind Funktionseinschränkungen und –verlust, hervorgerufen durch eine massive Akkumula-tion von Triglyzeriden in den Hepatozyten, Entzündungsprozesse sowie einem fibro-tischen Umbau der Leber. Im fortgeschritten Stadium wird eine Lebertransplantation unausweichlich, die jedoch aufgrund des zunehmenden Mangels an Spenderorganen oft nicht möglich ist. Eine Alternative bietet die Transplantation mesenchymaler Stammzellen (MSC). MSC können in vitro in leberzellähnliche Zellen differenziert wer-den und weisen dabei essentielle hepatozytäre Eigenschaften auf, wodurch sie als möglicher Ersatz bzw. als Überbrückungstherapie bis zur Lebertransplantation in Frage kommen. Die vorliegende Arbeit beschäftigte sich mit dieser Fragestellung. Dazu wur-de ein Tiermodell der NASH mittels Methionin-Cholin-defizienter Diät (MCD-Diät) etab-liert und die Transplantation von hepatozytär differenzierten MSC durchgeführt. An-hand spezifischer zellulärer und biochemischer Marker der NASH konnte die Wirkung des Zelltransplantats auf die Empfängerleber analysiert werden. Es hat sich gezeigt, dass die MSC einen anti-inflammatorischen, anti-fibrotischen und pro-proliferativen Einfluss auf das Empfängerparenchym hatten und somit zur Verbesserung der Symptomatik der NASH beitrugen.

Methylation of histone 3 (H3) at lysine 36 (K36) has been implicated in several biological processes, such as DNA replication, DNA repair, and transcription. To date, at least eight distinct mammalian enzymes have been described to methylate H3K36 in vitro and/or in vivo. In this work, Set2, Nsd1, and Nsd3 Venus tagged proteins were successfully expressed in mouse embryonic stem cells and, then, analyzed by confocal microscopy, mass spectrometry (MS), and chromatin immunoprecipitation sequencing (ChIP-seq). MS analysis revealed that Setd2, Nsd1, and Nsd3 do not associate in protein complexes with each other. Setd2 was associated with RNA polymerase II subunits and two transcription elongation factors (Supt5 and Supt6), whereas Nsd1 associated with the transcription factor Zfx. In contrast, Nsd3 interacted with multiple protein complexes including Kdm1b and Brd4 complexes. Interestingly, Nsd1 and Zfx seem to be bound to chromatin during cell division. ChIP-seq analysis of the H3K36 methyltransferases showed different binding profiles at transcribed genes: Nsd1 binds near the transcription start site (TSS), Setd2 loading starts near the TSS and spreads along the gene body, while, Nsd3 is preferentially enriched at the 5’ and 3’ gene regions. Sequential deletion of PWWP and zinger-finger like domains was achieved to study any possible changes in Nsd1 and Nsd3 function. Deletion of either PHD1-4 or PHD5/C5HCH domains decreased Nsd1 recruitment to chromatin. Particularly, the PHD5/C5HCH were identified as the protein-protein interface for Zfx interaction. In agreement, Zfx knockdown also decreased Nsd1 deposition at the Oct4 and Tcl1 promoter regions. Furthermore, Nsd1 depletion reduced bulk histone H3K36me2 and histone H3K36me3 loading at the coding regions of Oct4, Rif1, Brd2, and Ccnd1. In addition, Nsd1 knockdown led to an increased Zfx deposition at promoters. Our findings suggest Zfx recruits Nsd1 to its target loci, whereas Nsd1 regulates Zfx chromatin release and further contributes to transcription regulation through its H3K36 dimethylase activity. On the other hand, loss of Nsd3’s PHD5/C5HCH or PWWP domains decreased Nsd3 binding to DNA. In addition, we demonstrate that Nsd3 is recruited to target genes in a Brd4-dependent manner. Herein, we provided further insights on how H3K36 methyltransferases are regulated, and how they contribute to changes in the epigenetic landscape in mouse embryonic stem cells.fi

Microtubules are tubular polymers that are involved in a variety of cellular processes such as cell movement, mitosis and intracellular transport. The dynamic behavior of microtubules makes this possible because all of these processes require quick responses. Embryonic stem (ES) cells were first isolated from mouse embryos and they have two unique characteristics; they can be kept undifferentiated for many passages with a stable karyotype and they can be differentiated into any type of cells under appropriate conditions. The pluripotency of ES cells, their ease of manipulation in culture, and their ability to contribute to the mouse germ-line provides us a model of differentiation both in vitro and in vivo. In my thesis I focused on the cell division and neuronal differentiation of ES cells and developed two methods to understand the effects of microtubule dynamics in spindle assembly and chromosome segregation and to reveal the roles of different Microtubule Associated Proteins (MAPs) in the neuronal morphology formation. In the first part, we developed a live-cell imaging method for ES cells to visualize, track and analyze the single cell behavior in a cell population over a time period. So far many techniques have been adapted and combined for imaging of cell lines, mainly for the cancer or immortalized ones. However, because ES cells are very prone to apoptosis, tend to form spheres and hard to stably label, it is quite tricky to image them in culture conditions. In our system, we combined the BAC-based gene expression with wide-field deconvolution microscopy for ES cells that are plated onto the laminin-511 coated surface and kept in CO2 independent culture conditions. This combined technique does not interfere with the growth of cells and keeps them healthy up to 24 hours on the microscope stage. In the second part, we analyzed the effects of MAPs chTOG, EB1, Kif18A and MCAK in the overall spindle morphology and mitotic progression in mES cells. For this purpose, we utilized our stable TUBB-GFP and H2A-GFP cell lines along with our live-cell imaging set-up to reveal the effects of the above-mentioned proteins and the interplay among each other. By using RNAi method we either single or co-depleted the genes by siRNAs and measured the spindle length and width in RNAi conditions. We further analyzed the mitotic progression in H2A-GFP cell line in terms of the metaphase timing and the percentage of chromosome segregation errors. Our results showed that, EB1 depletion did not cause any significant changes in the overall spindle morphology or in the metaphase timing. However, the co-depletion of EB1 with chTOG partially rescued the sichTOG specific mini-spindle phenotype. siKif18A produced longer spindles without any change in the spindle width. Surprisingly, the co-depletion of antagonistic chTOG and Kif18A proteins had additive effects on the spindle dynamics and on mitotic progression in a way that spindle assembly was severely disrupted by the absence of these two proteins and as a result of this, both metaphase timing and chromosome missegregation levels increased significantly. These results overall indicate that MAPs have important roles in the regulation of dynamic instability and these proteins have an interplay among each other to be able to control the morphology of the spindle as well as the correct segregation of chromosomes into daughter cells. In the last part, I will introduce you a new ES cell based differentiation and morphology model, which brings the advantages of high resolution imaging capacity, control over development and easy genetic manipulation and culturing. We have generated Tet-induced shRNA cell lines against chTOG, Kif18A and MCAK, which are also stably expressing TUBB-GFP. These labeled cells were mixed with unlabeled wild-type mES cells before differentiation at 1:1000 ratio and then they were differentiated into mouse cortical cells and spinal motor neurons. Our results showed that, all of the three genes could be successfully knocked-down by shRNA after 48 hours of Tet induction. After mixing the labeled and unlabeled cells, single neurons could be imaged at high resolution and their skeletons could be generated afterwards. The RNAi studies in shchTOG cell line showed that, the knock-down of this gene in early differentiation interferes with the neuronal differentiation.

An intact and fully functional innate immune system is critical in the defense against pathogens. Indeed, during systemic infection, the ability of the organism to cope with the increased demand for phagocytes depends heavily on sufficient replenishment of mature myeloid cells. This process, designated emergency or demand-adapted myelopoiesis, requires the activation of hematopoietic progenitors in the bone marrow (BM), resulting in their proliferation and differentiation toward the myeloid lineage. Failure of BM progenitors to adapt to the enhanced need for mature cells in the periphery can be life-threatening, as indicated by the detrimental effect of chemotherapy-induced myelosuppression on the outcome of systemic infection. Recent advances demonstrate an important role of not only committed myeloid progenitors but also of hematopoietic stem cells (HSCs) in emergency myelopoiesis. In this regard, pathogen-derived products (e.g., Toll-like receptor ligands) activate HSC differentiation towards the myeloid lineage, either directly or indirectly, by inducing the production of inflammatory mediators (e.g., cytokines and growth factors) by hematopoietic and nonhematopoietic cell populations. The inflammatory mediators driving demand-adapted myelopoiesis target not only HSCs but also HSC-supportive cell populations, collectively known as the HSC niche, the microenvironment where HSCs reside. In this review, we discuss recent findings that have further elucidated the mechanisms that drive emergency myelopoiesis, focusing on the interactions of HSCs with their BM microenvironment.

The bone marrow (BM) niche provides hematopoietic stem (HSC) and progenitor cells with many exogenous cues that tightly regulate homeostasis. These cues orchestrate cellular decisions, which are difficult to dissect and analyze in vivo. This thesis introduces a novel in vitro platform that permits systematic studies of BM-relevant factors that regulate homeostasis. Specifically, the role of 3D patterned adhesion ligands and soluble cytokines were studied in a combinatorial fashion. Analysis of human HSC differentiation and proliferation at both population and single cell level showed synergistic and antagonistic effects of adhesion- and cytokine-related signals. Those effects were dependent on the cytokine concentration and the distribution and number of adhesion ligands. The aim of this thesis was to model the in vivo bone marrow with its porous 3D structure and different sized niche compartments using a microcavity culture carrier. The developed culture system presented extracellular matrix (ECM) adhesion ligands to the HSCs in various defined dimensions ranging from single- to multi-cell capacity. The 3D open well geometry of the microcavity carriers also allowed HSCs to freely explore different scenarios including homing, migration, adhesion, or suspension. Furthermore, the developed setup offered straightforward accessibility to analytical methods like cytometry and quantitative microscopy. Single cell analysis of adherent HSCs showed decreased DNA synthesis and higher levels of stem cell marker expression within single cell microcavities under low cytokine conditions . This effect was reflected in a decline of proliferation and differentiation with decreasing microcavity size. When the cytokine concentration was increased2 beyond physiological levels the inhibitory effect on proliferation and differentiation due to single-cell-microcavity adherence was diminished. This result highlighted the fine balance between adhesion related and soluble cues regulating HSC fate. Within small microcavities more adhesion related receptors were engaged due to the 3D character of the culture carrier compared to multi-cell wells or conventional 2D cell culture plates. This study demonstrated that adhesion-related signal activation leads to reduced proliferation and differentiation. This geometry-based effect could be reversed by increased cytokine supplementation in the culture media. For plane substrates, HSCs attachment to fibronectin or heparin initiated early cell cycle entry compared to non-adherent cells during the initial 24h. Cytokine supplemented media favored integrin activation that induced fast adhesion, ultimately leading to early cell cycle activation. However, after prolonged cell culture the system balanced itself with a lower cycling rate of adherent versus non-adherent HSCs. Furthermore, HSCs within the 3-dimensionality of the microcavities cycled less than 2D adherent cells. These findings additionally supported the above stated idea of limited HSC proliferation as a consequence of more adhesion-related signals overwriting cytokine driven expansion. To complement the various in vitro studies, an in vivo repopulation study was performed. Cultured HSCs derived from single cell microcavities outperformed freshly isolated HSCs in a competitive repopulation assay, indicating that carefully engineered substrates are capable of preserving stem cell potential. Overall the reported findings provide a promising in vitro culture strategy that allows the stem cell field to gain a better understanding of the impact of distinct exogenous signals on human HSCs, which discloses new concepts for the wide scientific community working towards tissue engineering and regenerative medicine.:Kurzbeschreibung 4 Abstract 6 1 Introduction 8 1.1 Motivation 8 1.2 Objective 8 2 Basics 10 2.1 Stem Cells and their Role in Life 10 Stem Cells and their Niches 12 2.1.1 Hematopoietic Stem Cells 12 2.1.2 Hematopoietic Stem Cell Niche 14 2.1.3 The ECM Relevancy 16 2.1.4 HSC Relevant Cytokines 19 2.2 Cell Culture Scaffolds 21 2.2.1 General 2D, 3D 21 2.2.2 Substrate Engineering 22 2.2.3 Co-Culture versus the Artificial 3D Niche 23 3 Materials and Methods 25 3.1 Chemicals, Reagents and Equipment 25 3.2 Wafer Design and Surface Functionalization 29 3.3 Cell Culture and Analysis 31 3.3.1 HSC Culture in ECM-functionalized Microcavities 32 3.4 Surface Passivation 33 3.5 Mouse Bone Marrow Preparation 35 4 Results and Discussion 37 4.1 Scaffold Design and Preparation 37 4.1.1 Surface Characterization 37 4.1.2 Surface Passivation 39 Approaches for Surface Passivation 39 Efficiency of Surface Passivation 39 4.1.3 Redesigned Microcavities 43 4.2 Summarized Discussion of the Surface Passivation 44 4.3 HSC Culture inside Microcavities 45 4.3.1 HSC-ECM Interaction Reduces Proliferation 45 4.3.2 Population-wide Proliferation and Differentiation of Spatially Constrained HSCs . … 46 HSCs within Redesigned Microcavities 48 4.3.3 Colony-forming Ability of Microcavity Cultures 50 4.4 Single Cell Analysis of Differentiation 52 4.5 Cell Cycling Dependency on Cytokine Level 53 4.5.1 Plane Surfaces 54 4.5.2 Microcavities Reduce Cycling Frequency 57 4.6 Mice Repopulation of Microcavity Cultured HSCs 58 4.7 Summarized Discussion of the HSC–ECM Relation 60 4.8 Future Prospects 62 5 Summary 63 References 64 Figure Legend 73 Tables 73 Theses 74 6 Appendices I 6.1 FACS Principle I 6.1.1 HSC Staining for CD Marker and Cell Cycle Kinetics I 6.1.2 Apoptosis Test II 6.2 Differentiation and Proliferation on Redesigned Microcavities III 6.3 Colony-forming Capability of Microcavity Cultured Cells IV 6.4 Effect of Trypsin on HSC Properties in Long Term Culture IV 6.5 Surface Functionalization with SCF V 6.5.1 Analysis of the HSCs Grown on Immobilized SCF VI 6.5.2 SCF Immobilization and its Kinetics VII 6.5.3 c-kit Expression Kinetics and HSC Differentiation VIII Short Discussion on the Growth Factor Immobilization IX Publications X Posters X Proceedings XI Talks XI Patents XI Papers XI Awards XI 7 Danksagung: XII Selbstständigkeitserklärung: XIII / Die Homöostase der Hämatopoietischen Stamm- und Vorläuferzellen (HSC) in der Knochenmark Nische wird von einer Vielzahl exogener Faktoren gezielt reguliert. Diese Faktoren orchestrieren intrazelluläre Vorgänge, deren in vivo Analyse kompliziert ist. Die vorliegende These widmet sich einem neuen biotechnologischen Ansatz, der systematische Studien von Knochenmark-relevanten Faktoren ermöglicht. Im Speziellen wurde die Rolle 3D-präsentierter Zell Adhäsionsliganden in Kombination mit verschiedenen Konzentrationen löslicher Zytokine untersucht. Die Auswertung der Proliferation und Differenzierung von humanen HSC auf Einzelzell- und Populationsebene offenbarte die synergistischen und antagonistischen Effekte von Adhäsions- und Zytokinsignalen in ihrer Abhängigkeit von der Verteilung und der Anzahl von Adhäsionsliganden sowie der Zytokinkonzentration. Um die poröse Struktur des Knochenmarks in vivo-ähnlich darzustellen, wurde eine Zellkultur Plattform mit Mikrokavitäten verschiedenster Dimensionen von Multi- bis Einzelzellgröße entwickelt und mit Molekülen der extrazellulären Matrix beschichtet. Die Vorteile dieser Plattform liegen in der offenen 3D-Geometrie dieses mikrokavitäten Kultursystems, die den Zellen ermöglichte verschiedene Wachstumsbedingungen bezüglich Homing, Migration, Adhäsion oder Suspension frei zu erkunden. Das leicht zugängliche Setup eignete sich zudem hervorragend für die zytometrische Analyse der Zellen oder die quantitative Mikroskopie. Die Einzelzellanalyse adhärenter HSC ergab eine Reduktion von DNA Synthese und eine höhere Expression von Stammzelloberflächenfaktoren innerhalb der Einzelzell-Mikrokavitäten bei niedrigen Zytokinkonzentrationen . Dieser Effekt spiegelte sich auch auf Populationsebene in verminderter Proliferation und Differenzierung mit abnehmender Größe der Mikrokavitäten wider. Wurde die Zytokinkonzentration jedoch weit über physiologische Bedingungen erhöht, verminderte sich der Effekt (reduzierte DNA Synthese und höhere Stammzellfaktorexpression) beschrieben für die Einzelzellmikrokavitäten. Dieses Ergebnis verdeutlicht die empfindliche intrazelluläre Balance, vermittelt durch Adhäsionsignale und löslichen Faktoren, die das Verhalten von HSCs regulieren. Aufgrund des 3D-Charakters des Zellkulturträgers wurden innerhalb kleiner Mikrokavitäten mehr Adhäsionsrezeptoren ringsum die Zelle aktiviert. Dieser Vorteil gegenüber den Multizellkavitäten oder der herkömmlichen 2D–Zellkultur ermöglichte eine hohe Anzahl adhäsionsvermittelter Signale mit entsprechend höherer Proliferations-inhibitorischer Wirkung. Je höher die Konzentration der Zytokine war, desto stärker erfolgte die Stimulation der Proliferation und Differenzierung. Auf 2D Substraten, initiierte Adhäsion zu Fibronektin und Heparin innerhalb der ersten 24h einen frühen Zell-Zyklus-Start im Gegensatz zu nicht adhärenten Zellen. Die Zytokine im Zellmedium förderten die Integrin Aktivierung, was zu einer schnellen Zelladhäsion führte. Die Adhäsionsrezeptoren wiederum kooperieren mit Zytokinrezeptoren im Zellinneren und begünstigten damit einen zeitigeren Zell-Zyklus- Start. Allerdings stellte sich danach ein Gleichgewicht im Kultursystem ein, wobei weniger adhärente Zellen als nicht-adhärente Zellen den Zellzyklus durchliefen. Des Weiteren war die Zellzyklusrate innerhalb von 3D Mikrokavitäten niedriger verglichen mit herkömmlichen 2D Substraten. Diese Ergebnisse bestätigen ferner obenstehende These, dass Zytokin-induzierte Zellexpansion durch erhöhte Zelladhäsions-vermittelte Signale überschrieben wird. Um die in vitro Studien zu komplettieren wurde ein in vivo Repopulationsversuch durchgeführt. HSC kultiviert auf Einzel-Zell-Mikrokavitäten übertrafen frisch isolierte Konkurrenz-Zellen in einem kompetitiven Repopulationsversuch. Dieses erste Ergebnis zeigt, dass sich der Zellgröße entsprechende Biomaterialien für die erfolgreiche Stammzell-Kultur eignen. Die Ergebnisse dieser Arbeit bieten eine vielversprechende in vitro Zellkulturstrategie, die ein besseres Verständnis der Einflüsse von exogenen Signalen auf HSC erlaubt und damit eine Grundlage für neue Erkenntnisse in Richtung erfolgreicheres Tissue Engineering und klinische Anwendungen im Bereich der regenerativen Medizin bildet.:Kurzbeschreibung 4 Abstract 6 1 Introduction 8 1.1 Motivation 8 1.2 Objective 8 2 Basics 10 2.1 Stem Cells and their Role in Life 10 Stem Cells and their Niches 12 2.1.1 Hematopoietic Stem Cells 12 2.1.2 Hematopoietic Stem Cell Niche 14 2.1.3 The ECM Relevancy 16 2.1.4 HSC Relevant Cytokines 19 2.2 Cell Culture Scaffolds 21 2.2.1 General 2D, 3D 21 2.2.2 Substrate Engineering 22 2.2.3 Co-Culture versus the Artificial 3D Niche 23 3 Materials and Methods 25 3.1 Chemicals, Reagents and Equipment 25 3.2 Wafer Design and Surface Functionalization 29 3.3 Cell Culture and Analysis 31 3.3.1 HSC Culture in ECM-functionalized Microcavities 32 3.4 Surface Passivation 33 3.5 Mouse Bone Marrow Preparation 35 4 Results and Discussion 37 4.1 Scaffold Design and Preparation 37 4.1.1 Surface Characterization 37 4.1.2 Surface Passivation 39 Approaches for Surface Passivation 39 Efficiency of Surface Passivation 39 4.1.3 Redesigned Microcavities 43 4.2 Summarized Discussion of the Surface Passivation 44 4.3 HSC Culture inside Microcavities 45 4.3.1 HSC-ECM Interaction Reduces Proliferation 45 4.3.2 Population-wide Proliferation and Differentiation of Spatially Constrained HSCs . … 46 HSCs within Redesigned Microcavities 48 4.3.3 Colony-forming Ability of Microcavity Cultures 50 4.4 Single Cell Analysis of Differentiation 52 4.5 Cell Cycling Dependency on Cytokine Level 53 4.5.1 Plane Surfaces 54 4.5.2 Microcavities Reduce Cycling Frequency 57 4.6 Mice Repopulation of Microcavity Cultured HSCs 58 4.7 Summarized Discussion of the HSC–ECM Relation 60 4.8 Future Prospects 62 5 Summary 63 References 64 Figure Legend 73 Tables 73 Theses 74 6 Appendices I 6.1 FACS Principle I 6.1.1 HSC Staining for CD Marker and Cell Cycle Kinetics I 6.1.2 Apoptosis Test II 6.2 Differentiation and Proliferation on Redesigned Microcavities III 6.3 Colony-forming Capability of Microcavity Cultured Cells IV 6.4 Effect of Trypsin on HSC Properties in Long Term Culture IV 6.5 Surface Functionalization with SCF V 6.5.1 Analysis of the HSCs Grown on Immobilized SCF VI 6.5.2 SCF Immobilization and its Kinetics VII 6.5.3 c-kit Expression Kinetics and HSC Differentiation VIII Short Discussion on the Growth Factor Immobilization IX Publications X Posters X Proceedings XI Talks XI Patents XI Papers XI Awards XI 7 Danksagung: XII Selbstständigkeitserklärung: XIII