Hematopoietic Stem Cell Differentiation inside Extracellular Matrix functionalized Microcavities

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

Private and Collaborative Kaplan-Meier Estimators

Kaplan-Meier estimators capture the survival behavior of a cohort. They are one of the key statistics in survival analysis. As with any estimator, they become more accurate in presence of larger datasets. This motivates multiple data holders to share their data in order to calculate a more accurate Kaplan-Meier estimator. However, these survival datasets often contain sensitive information of individuals and it is the responsibility of the data holders to protect their data, thus a naive sharing of data is often not viable. In this work, we propose two novel differentially private schemes that are facilitated by our novel synthetic dataset generation method. Based on these scheme we propose various paths that allow a joint estimation of the Kaplan-Meier curves with strict privacy guarantees. Our contribution includes a taxonomy of methods for this task and an extensive experimental exploration and evaluation based on this structure. We show that we can construct a joint, global Kaplan-Meier estimator which satisfies very tight privacy guarantees and with no statistically-significant utility loss compared to the non-private centralized setting

Discovery of the cancer stem cell related determinants of radioresistance

AbstractTumors are known to be heterogeneous containing a dynamic mixture of phenotypically and functionally different tumor cells. The two concepts attempting to explain the origin of intratumor heterogeneity are the cancer stem cell hypothesis and the clonal evolution model. The stochastic model argues that tumors are biologically homogenous and all cancer cells within the tumor have equal ability to propagate the tumor growth depending on continuing mutations and selective pressure. By contrast, the stem cells model suggests that cancer heterogeneity is due to the hierarchy that originates from a small population of cancer stem cells (CSCs) which are biologically distinct from the bulk tumor and possesses self-renewal, tumorigenic and multilineage potential. Although these two hypotheses have been discussed for a long time as mutually exclusive explanations of tumor heterogeneity, they are easily reconciled serving as a driving force of cancer evolution and diversity. Recent discovery of the cancer cell plasticity and heterogeneity makes the CSC population a moving target that could be hard to track and eradicate. Understanding the signaling mechanisms regulating CSCs during the course of cancer treatment can be indispensable for the optimization of current treatment strategies

An epigenetic reprogramming strategy to re-sensitize radioresistant prostate cancer cells

Radiotherapy is a mainstay of curative prostate cancer treatment, but risks of recurrence after treatment remain significant in locally advanced disease. Given that tumor relapse can be attributed to a population of cancer stem cells (CSC) that survives radiotherapy, analysis of this cell population might illuminate tactics to personalize treatment. However, this direction remains challenging given the plastic nature of prostate cancers following treatment. We show here that irradiating prostate cancer cells stimulates a durable upregulation of stem cell markers that epigenetically reprogram these cells. In both tumorigenic and radioresistant cell populations, a phenotypic switch occurred during a course of radiotherapy that was associated with stable genetic and epigenetic changes. Specifically, we found that irradiation triggered histone H3 methylation at the promoter of the CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), stimulating its gene transcription. Inhibiting this methylation event triggered apoptosis, promoted radiosensitization, and hindered tumorigenicity of radioresistant prostate cancer cells. Overall, our results suggest that epigenetic therapies may restore the cytotoxic effects of irradiation in radioresistant CSC populations

DNA-Methylome based Tumor Hypoxia Classifier Identifies HPV-negative Head & Neck Cancer Patients at Risk for Locoregional Recurrence After Primary Radiochemotherapy

BACKGROUND Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in Head and Neck Squamous Cell Carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia. METHODS A DNA methylome-based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA-HNSCC cohort based on matched assignments using gene expression-based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of Human Papilloma Virus (HPV)-negative HNSCC patients treated with primary radiochemotherapy (RCHT). RESULTS While hypoxia-GSEs failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (LR, HR=4.3, p=0.001) and overall survival (OS, HR=2.34, p=0.03) but not distant metastasis (DM) after RCHT in the both cohorts. Hypoxia-M status was inversely associated with CD8 T-cells infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR=1.83, p=0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status. CONCLUSIONS Our findings highlight an unexplored avenue for DNA Methylation-based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors. TRIAL REGISTRATION Retrospective observational study from the German Cancer Consortium (DKTK-ROG), not interventional

Glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA): a molecularly distinct brain tumor type with recurrent NTRK gene fusions

Glioneuronal tumors are a heterogenous group of CNS neoplasms that can be challenging to accurately diagnose. Molecular methods are highly useful in classifying these tumors-distinguishing precise classes from their histological mimics and identifying previously unrecognized types of tumors. Using an unsupervised visualization approach of DNA methylation data, we identified a novel group of tumors (n = 20) that formed a cluster separate from all established CNS tumor types. Molecular analyses revealed ATRX alterations (in 16/16 cases by DNA sequencing and/or immunohistochemistry) as well as potentially targetable gene fusions involving receptor tyrosine-kinases (RTK; mostly NTRK1-3) in all of these tumors (16/16; 100%). In addition, copy number profiling showed homozygous deletions of CDKN2A/B in 55% of cases. Histological and immunohistochemical investigations revealed glioneuronal tumors with isomorphic, round and often condensed nuclei, perinuclear clearing, high mitotic activity and microvascular proliferation. Tumors were mainly located supratentorially (84%) and occurred in patients with a median age of 19 years. Survival data were limited (n = 18) but point towards a more aggressive biology as compared to other glioneuronal tumors (median progression-free survival 12.5 months). Given their molecular characteristics in addition to anaplastic features, we suggest the term glioneuronal tumor with ATRX alteration, kinase fusion and anaplastic features (GTAKA) to describe these tumors. In summary, our findings highlight a novel type of glioneuronal tumor driven by different RTK fusions accompanied by recurrent alterations in ATRX and homozygous deletions of CDKN2A/B. Targeted approaches such as NTRK inhibition might represent a therapeutic option for patients suffering from these tumors

Expression of the transcription factor Hes3 in the mouse and human ocular surface, and in pterygium

Purpose: In this work we examined the presence of the neural stem cell biomarker Hairy and Enhancer of Split 3 (Hes3) in the anterior eye segment and in the aberrant growth condition of the conjunctiva pterygium. Further, we studied the response of Hes3 to irradiation. Materials and methods: Adult mouse and human corneoscleral junction and conjunctiva, as well as human pterygium were prepared for immunohistochemical detection of Hes3 and other markers. Total body irradiation was used to study the changes in the pattern of Hes3 expression. Results: The adult rodent and human eye as well as pterygium, contain a population of cells expressing Hes3. In the human eye, Hes3-expressing (Hes3+) cells are found predominantly in the subconjunctival space spanning over the limbus where they physically associate with blood vessels. The cytoarchitecture of Hes3 + cells is similar to those previously observed in the adult central nervous system. Furthermore, irradiation reduces the number of Hes3 + cells in the subconjunctival space. In contrast, irradiation strongly promotes the nuclear localization of Hes3 in the ciliary body epithelium. Conclusions: Our results suggest that a recently identified signal transduction pathway that regulates neural stem cells and glioblastoma cancer stem cells also operates in the ocular surface, ciliary body, and in pterygium

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements