Gastrulation leads to the formation of the embryonic germ layers, ectoderm, mesoderm and endoderm, and is the first key morphogenetic process that occurs in development. Gastrulation provides a unique developmental assay system in which to study cellular movements and rearrangements in vivo. The different cell movements occurring during gastrulation take place in a highly coordinated spatial and temporal manner, indicating that they must be controlled by a complex interplay of morphogenetic and inductive events. Generally, cell movement constitutes a highly integrated program of different cellular behaviors including sensing, polarization, cytoskeletal reorganization, and changes in adhesion and cell shape. During migration, these different behaviors require a continuous regulation and feedback control to direct and coordinate them. In this work, we analyze the cellular and molecular mechanisms underlying the different types of cell behaviors during gastrulation in zebrafish. Specifically, we focus on the role of the adhesive and mechanical properties of germ layer progenitors in the regulation of gastrulation movements. In the first part of the project, we investigated the role of the adhesive and mechanical properties of the different germ layer progenitor cell types for germ layer separation and stratification. In the second part of this study, we applied the same methodology to determine the function of germ layer progenitor cell adhesion in collective cell migration. Tissue organization is thought to depend on the adhesive and mechanical properties of the constituent cells. However, it has been difficult to determine the precise contribution of these different properties due to the lack of tools to measure them. Here we use atomic force microscopy (AFM) to quantify the adhesive and mechanical properties of the different germ layer progenitor cell types. Applying this methodology, we demonstrate that mesoderm and endoderm progenitors are more adhesive than ectoderm cells and that E-cadherin is the main adhesion molecule regulating this differential adhesion. In contrast, ectoderm progenitors exhibit a higher actomyosin-dependent cell cortex tension than mesoderm and endoderm progenitors. Combining these data with tissue self-assembly in vitro and in vivo, we provide evidence that the combinatorial activities of cell adhesion and cell cortex tension direct germ layer separation and stratification. It has been hypothesized that the directionality of cell movement during collective migration results from a collective property. Using a single cell transplantation assay, we show that individual progenitor cells are capable of normal directed migration when moving as single cells, but require cell-cell adhesion to participate in coordinated and directed migration when moving collectively. These findings contribute to the understanding of the gastrulation process. Cell-cell adhesion is required for collective germ layer progenitor cell migration, and cell cortex tension is critical for germ layer separation and stratification. However, many questions still have to be solved. Future studies will have to explore the interaction between the adhesive and mechanical progenitor cell properties, as well as the role of these properties for cell protrusion formation, cell polarization, interaction with extracellular matrix, and their regulation by different signaling pathways.

No description available.

Malgré l’efficacité thérapeutique avérée des agents anti-angiogéniques et des agents anti-vasculaires (VDA), le mécanisme d’action précis des stratégies ciblant les vaisseaux sanguins tumoraux, les raisons de leur efficacité ainsi que les mécanismes de résistance à ces drogues sont encore mal compris. Il est rapidement apparu essentiel d’identifier des biomarqueurs capables de refléter l’angiogénèse tumorale ou les effets sur la vascularisation tumorale de ces traitements. Compte tenu de leur importance dans des pathologies vasculaires, les cellules endothéliales matures circulantes (CEC) et les progéniteurs endothéliaux circulants (CEP) ont d’emblée été pressenties comme des candidats intéressants pour être des biomarqueurs de réponse aux stratégies ciblant la vascularisation tumorale. Nous avons exploré l’intérêt de ces cellules en tant que biomarqueurs de l’angiogénèse dans des tumeurs pédiatriques, et leur rôle en tant que biomarqueurs de traitement par des agents anti-angiogéniques chez des sujets adultes atteints de cancer. Ces travaux ont mis en lumière l’intérêt des CEP et ont été à la source d’un travail plus « mécanistique » où nous avons étudié dans différents modèles murins le rôle des CEC et CEP dans le mécanisme d’action des agents anti-vasculaires et plus particulièrement le rôle fonctionnel des CEP dans la résistance à ces molécules. Par des stratégies d’association d’agents anti-angiogéniques aux VDA destinées à inhiber les CEP, nous montrons l’augmentation de l’activité anti-tumorale des VDA et offrons un rationnel mécanistique pour optimiser les schémas thérapeutiques actuels des traitements anti-vasculaires. Nos données apportent des arguments en faveur du rôle potentiel de ces cellules en tant que biomarqueurs de l’angiogénèse, des traitements anti-angiogéniques et de la résistance aux traitements anti-vasculaires. / Despite their therapeutic impact and clinical benefit, the mecanisms of action of anti-angiogenic agents and vascular disrupting agents (VDA), the reasons for their efficacy as well as the mechanisms underlying resistance to these drugs are not fully understood. Thus, identifying surrogate biomarkers of tumor angiogenesis and of the effects of these new therapeutic agents targeting tumor blood vessels has become a crucial objective. Because of their importance in vascular diseases, mature circulating endothelial cells (CEC) and circulating endothelial progenitor cells (CEP) were suggested to be potential candidate biomarkers of disease response and relapse to vascular targeting strategies. We investigated the role of these cells as biomarkers of tumor angiogenesis in pediatric solid tumors, as well as biomarkers of response to anti-angiogenic therapies in adult cancer patients. By revealing the particularly important role of CEP, these initial studies led to a more “mechanistic” study in which the cellular and molecular effects of a VDA were evaluated with regard to CEC and CEP in different mouse models; in particular, the “catalytic” role of CEP was explored as a mechanism of resistance to VDA. By combining anti-angiogenic agents aimed to inhibit CEP mobilized by the VDA, we demonstrate an increase in the anti-tumor activity of the VDA and offer a mechanistic rational to optimize VDA-based therapeutic strategies. Our data support the role of CEC and CEP as biomarkers of angiogenesis, of anti-angiogenic strategies and of resistance to vascular-disrupting therapies.

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

No description available.

Diabetes mellitus is a group of metabolic disorders that are characterized by chronic hyperglycaemia. There are currently over 460 million people living with diabetes and the incidence rate for the most common types is sharply on the rise. The hyperglycaemia and subsequently the majority of diabetic side-effects can be cured by ß-cell transplantation. There is a severe lack of donor tissue for clinical transplantations, hence ß cells derived from human pluripotent stem cells (hPSCs) offer an attractive option for obtaining the necessary cells for a ß-cell therapy. The current ß-cell differentiation protocols are lengthy, expensive and relatively inefficient. Therefore, it remains unrealistic to scale up production to obtain the necessary 1 billion cells per patient to achieve normoglycaemia. The exponential expansion of an intermediate pancreatic progenitor (PP) population would allow for significant reduction of the cost and time necessary for the production of ß-cells in vitro and, more importantly, it would allow to relatively easily obtain the number of cells necessary for clinical applications. Thus far, the reproducible expansion of suitable hPSC-derived PPs in a chemically-defined, feeder-free culture condition has not been reported. Our lab has found that a previously reported medium suitable for the temporary expansion of reprogrammed fibroblast-derived PPs could occasionally, in 20% of the cases, mediate expansion of PSC derived PPs. To elucidate the requirements for reproducible expansion, I analysed transcriptomic data from PSC-derived PPs before and following expansion. Several regulated signalling pathways were identified and from these data, I formulated nine candidate expansion conditions (C0-C8) to test. Five of these conditions were able to reproducibly expand hPSC-derived PPs. Of those five conditions, C6 was found to mediate the fastest expansion with a doubling time of 2.2 days, maintained a stable expression of the crucial bipotent trunk progenitor transcription factor (TF) markers PDX1, NKX6.1, SOX9 and FOXA2 and minimized the expression of the hepatic and intestinal markers AFP and CDX2. The C6 expanded PPs were able to further differentiate into pancreatic endocrine progenitors (PEPs) in three-dimensional (3-D) islet-sized clusters formed in micropatterned wells. Micropatterned wells offer the additional advantage of size-controlled, uniform clusters and low culture volumes compared to suspension culture bioreactors proposed for the large-scale production of such clusters, but the use of micropatterned wells has not been reported for such an application thus far. These PEPs showed strong upregulation of the crucial endocrine specific TF NGN3 and around 90% were positive for NEUROD1, a downstream effector of NGN3, as determined by flow cytometry analysis. The PEPs were subsequently differentiated into insulin (INS)-producing ß-cells at around 20% efficiency. The ß-cells were also strongly expressing functionality genes such as zinc transporter 8 (ZnT8), glucokinase (GCK), prohormone convertase 1/3 (PC1/3) and sulfonylurea receptor 1 (SUR1, a subunit of KATP channel) and around 10% of them were expressing the crucial maturation marker MAFA, as determined by flow cytometry analysis of ß-cells derived from the H1INS1-GFP/MAFA-mCHERRY double reporter line generated in the lab. In summary, I have established a feeder-free, chemically defined and ‘good manufacturing practice’ (GMP)-compatible culture condition for the exponential expansion of hPSC-derived PPs that allows for the production of ß-cells at a fraction of the cost of conventional differentiation protocols. Ongoing work is being done to further optimise the expansion conditions to completely eliminate hepatic and intestinal markers and to optimise the subsequent differentiation steps in micropatterned wells to achieve a high-efficiency differentiation towards functional ß-cells. / Diabetes Mellitus ist eine Gruppe metabolischer Krankheiten, die sich durch chronische Hyperglykämie charakterisiert. Es gibt zurzeit über 460 Millionen Menschen, die mit Diabetes leben und die Inzidenzrate der häufigsten Formen steigt dramatisch. Die Hyperglykämie und die Mehrheit der daraus resultierenden diabetischen Begleiterscheinungen können durch ß Zell Transplantation geheilt werden. Da es einen enormen Gewebespendermangel für klinische Transplantationen gibt, bieten von humanen pluripotenten Stammzellen (hPSCs) stammende ß Zellen eine attraktive Option, um die notwendigen Zellen für eine ß Zell Therapie zu erhalten. Die momentanen ß Zell Differenzierungsprotokolle sind langwierig, teuer und relativ ineffizient. Deshalb bleibt eine Produktionsvergrößerung zur Gewinnung der notwenigen 1 Milliarde Zellen pro Patient zum Erreichen einer Normoglykämie unrealistisch. Die exponentielle Expansion einer intermediären pankreatischen Vorläuferpopulation (PP) würde eine signifikante Kosten und Zeitreduktion zur Produktion von ß Zellen in vitro erlauben und, noch wichtiger, würde relativ einfach ermöglichen, die notwendigen Zellzahlen für klinische Anwendungen zu erzielen. Bisher wurde von einer reproduzierbaren Expansion geeigneter, von hPSC stammenden PPs in einer chemisch definierten Zellkulturbedingung, die frei von Feeder Zellen ist, nicht berichtet. Unser Labor fand heraus, dass ein im Vorfeld veröffentlichtes Medium, welches für die vorrübergehende Expansion von reprogrammierten, von Fibroblasten stammenden PPs geeignet ist, in 20% der Fälle die Expansion von aus hPSC gewonnenen PPs herbeiführen konnte. Um die Anforderungen für eine reproduzierbare Expansion zu eruieren, analysierte ich transkriptomische Daten von aus PSC gewonnenen PPs vor und nach erfolgter Expansion. Mehrere regulierte Signalwege wurden identifiziert und von diesen Daten formulierte ich neun zu testende Kandidatenexpansionsbedingungen (C0-C8). Fünf dieser Bedingungen ermöglichten es von PSC stammende PPs reproduzierbar zu expandieren. Von diesen fünf Bedingungen war C6 die, welche die schnellste Expansion mit einer Dopplungszeit von 2.2 Tagen herbeiführte, eine stabile Expression der essenziellen Marker von bipotenten Stammvorläuferzellen, PDX1, NKX6.1, SOX9 und FOXA2, aufrechterhielt und die Expression von den hepatischen und intestinalen Markern AFP und CDX2 minimierte. Den C6 expandierten PPs war es möglich, sich in pankreatisch endokrine Vorläufer (PEP) in dreidimensionalen (3 D), inselgroßen Kluster, welche in mikrogemusterten Wells geformt wurden, zu differenzieren. Mikrogemusterte Wells bieten den zusätzlichen Vorteil von größenkontrollierten, uniformen Kluster und niedrigen Kulturvolumina im Vergleich zu Suspensionskulturbioreaktoren, welche für die Großmengenproduktion solcher Kluster vorgeschlagen wurden. Jedoch wurde bisher über die Benutzung von mikrogemusterten Wells für solch eine Anwendung nicht berichtet. Diese PEPs zeigten eine starke Hochregulation des essenziellen, endokrin spezifischen Transkriptionsfaktors (TF) NGN3 und 90% waren positiv für NEUROD1, einem nachgelagerten Effektor von NGN3, was anhand der Analyse der Durchflusszytometrie bestimmt wurde. Darauffolgend wurden die PEPs mit einer Effizienz von etwa 20% in Insulin (INS) produzierende ß Zellen differenziert. Diese ß Zellen zeigten ebenfalls eine starke Expression von Funktionalitätsgenen wie ZnT8, GCK, PC1/3 und SUR1 und etwa 10% von ihnen exprimierten den essenziellen Maturationsmarker MAFA, wie es mittels Analyse der Durchflusszytometrie von aus einer INS-GFP/MAFA-mCHERRY Doppelreporterlinie gewonnenen ß Zellen bestimmt wurde. Zusammenfassend habe ich eine Kulturbedingung für die exponentielle Expansion von aus hPSC gewonnenen PPs etabliert, die frei von Feeder Zellen, chemisch definiert und kompatibel mit der Guten Herstellungspraxis ist, welche die Produktion von ß Zellen zu einem Bruchteil der Kosten konventioneller Differenzierungsprotokollen ermöglicht. Es wird weiterhin daran gearbeitet, die Expansionsbedingungen zu optimieren, um die hepatischen und intestinalen Marker komplett zu eliminieren und die darauffolgenden Differenziationsschritte in mikrogemusterten Wells zu verbessern, um letztendlich eine hocheffiziente Differenzierung zu funktionellen ß-Zellen zu erreichen.

Pancreatic β-cells have a central function in the regulation of glucose homeostasis by releasing the blood sugar-lowering hormone insulin. Disruption of this process results in diabetes, which has a tremendous impact on the quality of life and requires lifelong treatment. Elucidating the mechanisms of pancreatic progenitor cell differentiation into fully functional β-cells will contribute to identifying the underlying reasons for β-cell dysfunction and to finding a cure for diabetes. Aldh1b1 was identified by our research group as a regulator of pancreas development and β-cell functionality. Aldh1b1 is a mitochondrial enzyme, expressed in all embryonic pancreas progenitors. Its expression is switched off during the process of differentiation and is undetectable in differentiated cells. Functional inactivation of Aldh1b1 in the mouse leads to premature differentiation of progenitor cells in the embryo and dysfunctional β-cells in the adult. However, the enzymatic function of Aldh1b1 in pancreas progenitors and how it ultimately affects β-cell functionality remained to be elucidated. In this study, I analyzed the role of Aldh1b1 in the metabolism of embryonic pancreas progenitor cells and its impact on chromatin structure and gene expression in both, progenitors and postnatal β-cells. Flow cytometry analysis of freshly isolated Aldh1b1 null embryonic pancreas progenitors showed a significant increase in ROS levels as well as a significant decrease in mitochondrial mass, whereas the mitochondrial membrane potential was not affected. To elucidate the impact of Aldh1b1 on cellular metabolism, I conducted metabolic flux experiments and untargeted metabolomics studies using FACS-isolated embryonic pancreas progenitors expanded in a 3D spheroid culture. Analyses following metabolic labeling with either 13C6-Glucose or 13C2-Glutamine showed that the absence of Aldh1b1 lead to an increase of the reductive glutamine metabolism towards citrate, a reaction that channels carbon units into the acetyl-CoA biosynthesis. However, the ACLy-dependent flux towards acetyl-coA synthesis was reduced and this was consistent with reduced expression of ACLy as well as the citrate transporter SLC25a1. A decrease in cellular acetyl-CoA would reduce histone acetylation. Untargeted metabolomics showed an increase in the concentration of S-adenosyl-methionine, suggesting increased DNA and histone methylation. Consistent with these findings, ATAC-Seq analyses on freshly isolated pancreatic progenitors showed reduced chromatin accessibility at genes implicated in chromatin organization, protein acetylation and histone modification. Transcription motif analysis showed that the affected genomic sites were mainly associated with the binding of Klf/Sp and Nrf1 transcription factors. Transcriptome analyses displayed that the expression of genes implicated in progenitor differentiation, ECM organization and transcriptional regulation was affected. Furthermore, transcriptome analyses of early postnatal β-cells uncovered early signs of oxidative stress and increased proliferation, thus providing the basis to explain the β-cell phenotype in Aldh1b1 null mice. I then used organotypic cultures of embryonic pancreata to investigate the connection between high ROS levels and aberrant differentiation in the Aldh1b1 null pancreata. Reducing ROS levels using NAC enabled the reversal of the aberrant transcription factor expression and increased viability of Aldh1b1 null explants, thus identifying high ROS levels as a driving force in this process. To investigate how persisting Aldh1b1 expression would affect progenitor differentiation, I generated ROSA26LSLAldh1b1, an inducible constitutive Aldh1b1 expression line. Progenitors with continuous Aldh1b1 expression avoided the endocrine cell fate, underscoring the importance of timely Aldh1b1 downregulation in the course of β-cell differentiation. Altogether, my work provides strong evidence for the role of Aldh1b1 as a metabolic regulator in the process of progenitor cell differentiation and identifies a link between metabolism and gene regulation through chromatin accessibility during development. Aldh1b1 inactivity causes defects in embryonic progenitor cells as well as postnatal β-cells and could therefore contribute, as genetic risk factor, to the development of hyperglycemia and diabetes later in life. Comprehending the mechanisms underlying the process of pancreas progenitor differentiation as well as the origins of β cell dysfunction should assist in the design of novel therapeutic interventions for diabetes.

No description available.

No description available.

<p><strong>Rationale: </strong>Lung-homing of bone marrow (BM)-derived progenitor cells is associated with inflammatory and remodeling changes in asthma. Stromal cell derived factor-1α (SDF-1α) is a potent progenitor cell chemoattractant and its local production in the lung promotes lung homing of progenitor cells. The role of pro-inflammatory cytokines in promoting traffic of progenitor cells to the site of inflammation in asthma has not been investigated. The TH2 cytokines, interleukin (IL)-4 and IL-13, are key regulators of asthma pathology.</p> <p><strong>Objective: </strong>To investigate the role of IL-4 and IL-13 in modulating the trans-migrational responses of hemopoietic progenitor cells (HPC).</p> <p><strong>Methods: </strong>HPC were isolated from cord blood (CB) and peripheral blood (PB) and migrational and adhesive responses were assessed using transwell migration assays and adhesion to fibronectin-coated wells, respectively. Responding cells were enumerated by flow cytometry.</p> <p><strong>Results: </strong>IL-4 and IL-13 had no direct effect on progenitor cell migration. Pre-incubation with each of these cytokines primed SDF-1α stimulated migration of CB and PB-derived HPC (CD34+45+ cells) but not eosinophil-lineage committed progenitors (CD34+45+IL- 5Rα+ cells) or mature eosinophils to SDF-1α. For HPC, priming effects of IL-4 (0.1ng/ml) and IL-13 (0.1ng/ml) were detectable within 1hr and optimal at 18hr post- incubation and IL-4 was the more effective priming agent. Disruption of lipid rafts inhibited IL-4 priming of SDF-1α stimulated migration of HPC indicating that increased incorporation of CXCR4 into membrane lipid rafts mediates the cytokine primed migrational response of HPC. This was confirmed by confocal fluorescent microscopy.</p> <p><strong>Conclusions: </strong>IL-4 and IL-13 prime the migrational response of HPC to SDF-1α by enhancing the incorporation of CXCR4 into lipid rafts. The priming effect of these cytokines is specific to primitive HPC. These data suggest that increased local production of IL-4 and IL-13 within the lungs may promote increased SDF-1α mediated homing of BM-derived HPC to the airways in asthma.</p> / Master of Science in Medical Sciences (MSMS)